CN110885856B - Expression vector for controllably up-regulating ACE2 targeting prevention and treatment of hypoxic pulmonary hypertension - Google Patents

Abstract

The invention discloses an expression vector for controllably up-regulating ACE2 targeting prevention and treatment of low-oxygen pulmonary hypertension. The invention constructs an ACE2 expression vector driven by an HRE-enhanced Tie2 promoter, which is specifically positioned in vascular endothelial cells through the Tie2 promoter, and simultaneously, the HRE is specifically activated by using the expression of HIF-1 alpha only in hypoxic pulmonary vascular endothelial cells, thereby up-regulating the expression of ACE2. The invention discovers that the constructed expression vector not only has targeting property, but also can controllably and effectively up-regulate the expression of ACE2 in hypoxic pulmonary microvascular endothelial cells according to the degree of hypoxia so as to regulate and control pulmonary artery smooth muscle cells, realize hypoxia controllability and targeted inhibition of pulmonary artery contraction and reversal of pulmonary artery structural reconstruction, and provide effective means and strategies for preventing and treating diseases such as hypoxic pulmonary hypertension and the like.

Description

Controllable up-regulation of ACE2 expression vector for targeted prevention and treatment of hypoxic pulmonary hypertension

technical field

The invention relates to the field of applying gene recombination technology to treat hypoxic pulmonary hypertension, in particular to a method for constructing an expression vector of angiotensin-converting enzyme 2 (ACE2) driven by an HRE-enhanced Tie2 promoter.

Background technique

Hypoxic pulmonary hypertension (HPH) is a common link in many respiratory diseases, chronic altitude sickness and various diseases related to hypoxemia. Hypoxic pulmonary vasoconstriction caused by hypoxia is one of the important physiological responses of the body, and it is of great significance in maintaining the ventilation/blood flow ratio around the hypoxic alveoli, reducing functional shunts, and increasing blood oxygen saturation. However, long-term and extensive hypoxic pulmonary vasoconstriction can lead to remodeling of pulmonary vascular structure. The reconstruction of pulmonary vascular structure is a key factor that causes continuous increase in pulmonary artery pressure and promotes the occurrence and development of cor pulmonale. Therefore, specific relaxation of pulmonary arteries, inhibition or reversal of pulmonary vascular remodeling is the main goal of prevention and treatment of hypoxic pulmonary hypertension and its complications.

Studies have shown that the pulmonary renin-angiotensin (RAS) system is involved in the occurrence and development of hypoxic pulmonary hypertension. In hypoxic pulmonary hypertension, the main effector protein of RAS, angiotensin II (AngII), not only strongly constricts pulmonary blood vessels, but also significantly promotes the proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMCs), which is one of the important factors that cause the continuous increase of pulmonary vascular resistance. . Other members of RAS, such as renin and angiotensinogen, are also involved in the development of HPH in various ways. Therefore, in hypoxic pulmonary hypertension, abnormal function of RAS in the lung may be one of the important reasons for hypoxic pulmonary vasoconstriction and structural reconstruction. Recently discovered that ACE2, a new member of RAS, is a membrane protein composed of 805 amino acids, widely distributed in heart and lung vascular endothelial cells, vascular smooth muscle cells, lung epithelial cells, cardiomyocytes and gastrointestinal tract, Guwan, retina, uterus , placenta and other tissues. Its main function is to efficiently generate angiotensin-(1-7)[Angiotensin-(1-7),Ang-(1-7)]. ACE2 can degrade angiotensin I (Ang I) to Ang-(1-9), then Ang-(1-9) under the action of neutral endopeptidase and prolyl endopeptidase Generate Ang-(1-7). ACE2 can also directly enzymolyze angiotensin II (Ang II) to generate Ang-(1-7). Ang-(1-7) promotes vasodilation, anti-proliferation, and anti-hypertrophy by binding to its specific Mas receptor. By producing a large amount of Ang-(1-7), ACE2 can fine-tune the balance between vasoconstrictor active substances and vasodilation active substances in the body. In addition, ACE2 can also participate in maintaining the stability of the body's internal environment by acting on various active substances in the body. Therefore, increasing the expression of ACE2 in the pulmonary circulation will help to improve the dysfunction of RAS in the lungs, inhibit hypoxic pulmonary vasoconstriction and reverse the reconstruction of pulmonary vascular structure, and then play an effective role in preventing and treating hypoxic pulmonary hypertension.

However, the conventional route of administration lacks specificity, leading to the side effect of lowering systemic blood pressure. Moreover, long-term, single expansion of pulmonary vessels can also lead to venous blood doping and aggravate hypoxemia. How to make ACE2 specifically localize in the pulmonary blood vessels, and only increase the expression in the pulmonary blood vessels under hypoxia, and also realize the controllable regulation according to the degree of hypoxia, is the key point of prevention and treatment of hypoxic pulmonary hypertension. The essential.

Chinese patent CN101532027 (disclosure date: September 16, 2009) discloses a hypoxia-induced eukaryotic gene expression vector and its application. Through the transformation of pcDNA3.1, its enhancer is used as an hypoxia-induced enhancer Instead, a hypoxia-inducible gene expression vector was constructed; in order to test the function of the hypoxia-inducible gene expression vector, the cDNA of hVEGF165 was inserted into the vector, and the hypoxia-inducible eukaryotic gene expression vector p6HRE-hVEGF165 containing hVEGF165 was constructed, and the The vector was transferred into BHK cells, and the BHK cells expressing hVEGF were cultured in a hypoxic environment, and the content of hVEGF in the culture supernatant was detected by ELISA, and the results proved that the expression of hVEGF increased after induction of hypoxia; p6HRE-hVEGF165 The gene therapy for the ischemic disease of rabbit limbs can effectively promote the formation of new blood vessels and collateral circulation in the affected limbs, and restore the pressure of the tibial arteries of the affected limbs.

However, the pulmonary circulation vessels have significantly different characteristics from the systemic circulation vessels in the above-mentioned patents. Hypoxic contraction of pulmonary blood vessels occurs during short-term hypoxia to maintain the ventilation/blood flow ratio around the hypoxic alveoli, reduce functional shunts, and increase blood oxygen saturation; long-term hypoxia causes endothelin and serotonin vascular , endothelial growth factor (VEGF) and other vasoactive substances secrete excessively and then participate in pulmonary vascular remodeling, aggravating the degree of pulmonary ischemia and hypoxia, which is the pathophysiological feature of hypoxic pulmonary hypertension. Blood vessels in the systemic circulation obviously dilate when hypoxic, increasing blood flow to alleviate the symptoms of ischemia and hypoxia. Therefore, hypoxic pulmonary hypertension (HPH) requires more precise expression regulation according to the degree of hypoxia.

Contents of the invention

The purpose of the present invention is to provide an expression vector for controllable up-regulation of ACE2 for targeted prevention and treatment of hypoxic pulmonary hypertension.

To achieve the above object, the present invention adopts the following technical solutions:

A recombinant expression vector of ACE2 (HTSFCACE2 for short), comprising a hypoxia response element, a promoter, a signal peptide (Signal Peptide) sequence and an ACE2 coding sequence, wherein the hypoxia response element is selected from an HRE sequence, and the At the same time, the expression of ACE2 is specific to vascular endothelial cells by using the promoter.

Preferably, the number of repetitions of the HRE sequence is 1-100.

Preferably, the promoter is selected from the Tie2 gene promoter.

Preferably, the expression vector specifically includes 6×HRE sequence, Tie2 gene promoter sequence, signal peptide, hIgG1Fc fusion marker sequence and ACE2 coding sequence arranged in sequence.

Preferably, the expression vector is constructed on the backbone of the adeno-associated virus vector or the backbone of the eukaryotic expression vector.

The method for constructing the above-mentioned expression vector comprises the following steps: synthesizing 6×HRE sequence, Tie2 gene promoter sequence, signal peptide, hIgG1Fc fusion marker sequence and ACE2 coding sequence; cloning each synthetic sequence into the expression vector backbone (for example, pAAV- MCS), and the resulting recombinant plasmid was called HRE-Tie2-FC-ACE2 (HTSFCACE2 for short). A recombinant plasmid not containing the ACE2 coding sequence was used as a control plasmid, which was called HRE-Tie2-FC (abbreviated as HTSFC).

Use of the above expression vector (HTSFCACE2) in the preparation of medicines for preventing and/or treating hypoxic pulmonary hypertension and/or complications of hypoxic pulmonary hypertension.

Preferably, the expression vector has cell targeting, and under normoxia (21% O ₂ concentration) condition, ACE2 can be highly specifically expressed only in transfected pulmonary microvascular endothelial cells (PMVECs).

Preferably, the expression vector has the controllability of hypoxia (oxygen concentration lower than 21%, that is, hypoxia), and under the stimulation of different concentrations of hypoxia (10%, 5% and 1% O ₂ ), after transfection The expression of ACE2 in PMVECs cells and cell supernatant increased significantly, and the increase rate increased with the decrease of oxygen concentration.

Preferably, the expression vector has the effect of inhibiting the proliferation of PASMCs. For example, after PMVECs are transfected with HTSFCACE2, hypoxia stimulation with 10% oxygen concentration is given, and the PMVECs cell supernatant collected after continuing to culture for 24 hours is the PMVECs containing the expression vector HTSFCACE2 Hypoxic condition culture solution, the PMVECs hypoxic condition culture solution has obvious inhibitory effect on hypoxia-induced pulmonary artery smooth muscle cell (PASMCs) proliferation.

Preferably, the HTSFCACE2 expression vector packaged as an adeno-associated virus acts on hypoxic pulmonary hypertension rats in a one-time nasal drop, which can reduce the right ventricular pressure of the rats, reduce the degree of right ventricular hypertrophy, and inhibit the structural changes of the pulmonary arterioles , improve pulmonary artery structure reconstruction, and after the virus enters the rat body, the expression of ACE2 increases only in lung tissue, reduces the content of angiotensin II (AngII) in lung tissue, and increases angiotensin (1-7) [Ang(1 -7)] content, with tissue specificity and effectiveness.

Use of the above expression vector (HTSFCACE2) in the preparation of medicines for preventing and/or treating a series of diseases related to hypoxia and/or ischemia, such as diabetic gangrene, deep vein thrombosis, and ischemic heart disease.

The beneficial effects of the present invention are reflected in:

The present invention uses the specificity of promoter-driven expression localized in vascular endothelial cells, the hypoxic conditional expression of HRE and the characteristics of ACE2 to finely regulate the renin-angiotensin (RAS) system in the lung to construct a HRE-enhanced, promoter ( For example, the ACE2 expression vector driven by the Tie2 gene promoter) can enable ACE2 to be highly expressed in hypoxic PMVECs in a targeted, controllable and effective manner, thereby regulating PASMCs, inhibiting the contraction and structural reconstruction of pulmonary arteries, and preventing hypoxia. The role of oxygen pulmonary hypertension provides a new strategy and important experimental basis for the study of gene therapy for hypoxic pulmonary hypertension and its complications, and has a good application prospect.

Further, the present invention improves the tissue specificity of expression by using the Tie2 gene promoter (Tie2 promoter), solves the side effect of lowering systemic blood pressure caused by the existing eukaryotic expression vector promoter, and avoids long-term, single reduction of pulmonary vascular Stress-induced doping of venous blood and exacerbating the problem of hypoxemia in patients. At the same time, the controllability and accuracy of expression are improved.

Furthermore, the present invention ensures sufficient combination with subsequent sequences during hypoxia by optimizing the repeat times of the determined HRE sequence, and improves the controllability and accuracy of expression.

Further, the experiments of the present invention verified that the recombinant plasmid HTSFCACE2 can be highly expressed in hypoxic pulmonary artery endothelial cells, thereby regulating pulmonary artery smooth muscle cells, realizing controllable hypoxia, targeted inhibition of pulmonary artery contraction and reversal of pulmonary artery structural reconstruction. The recombinant plasmid is also applicable to a series of diseases related to hypoxia or/and ischemia, such as diabetic gangrene, deep vein thrombosis, and ischemic heart disease.

Description of drawings

Figure 1 is a schematic diagram of the structure of the recombinant plasmid HTSFCACE2 and the control plasmid.

Figure 2 is the result of enzyme digestion identification of the recombinant plasmid HTSFCACE2; lane M is marker, and lanes 1 and 2 are HTSFCACE2.

Figure 3 is the result of enzyme digestion identification of the control plasmid HTSFC; lane M is marker, and lanes 1 and 2 are HTSFC.

Figure 4 shows the expression changes of ACE2 in different types of cells transfected with HTSFCACE2 under normoxia (21% O ₂ ); among them: (A) and (B) are the schematic diagrams and quantitative diagrams of the expression of ACE2 in different types of cells, *P<0.05 vs.untransduced PMVECs cells, n=5; (C) is the change of ACE2 content in the supernatant of different types of cells, *P<0.05vs.untransduced PMVECs cells, n=5.

Figure 5 is the effect of recombinant plasmid HTSFCACE2 on the expression of ACE2 in PMVECs cells stimulated by normoxia (21% O ₂ ) and different degrees of hypoxia (10%, 5% and 1% O ₂ ); wherein: (A) and ( B) is the schematic diagram and quantification diagram of ACE2 expression in PMVECs cells, *P<0.05vs.untransduced PMVECs cells, n=5; (C) is the change of the increase rate of ACE2 expression in PMVECs cells, *P<0.05vs.Normoxia +transduced HTSFCACE2, n=5.

Figure 6 is the effect of recombinant plasmid HTSFCACE2 on the ACE2 content in the PMVECs cell supernatant stimulated by normoxia (21% O ₂ ) and different degrees of hypoxia (10%, 5% and 1% O ₂ ); wherein: (A) It is the change of ACE2 content in PMVECs cell supernatant, *P<0.05vs.untransduced PMVECs cells, n=5; (B) is the change of ACE2 content increase rate in PMVECs cell supernatant, *P<0.05vs.Normoxia +transduced HTSFCACE2, n=5.

Figure 7 is the effect of PMVECs hypoxic condition culture medium containing HTSFCACE2 on the proliferation of PASMCs stimulated by normoxia (21% O ₂ ) and different degrees of hypoxia (10%, 5% and 1% O ₂ ); wherein: (A ) is the effect of normoxia (21% O ₂ ) and hypoxia (10%, 5% and 1% O ₂ ) stimulation on the proliferation of PASMCs, *P<0.05vs.Normoxia group, n=5; ( B) is the effect of PMVECs hypoxic condition culture medium containing HTSFCACE2 on the proliferation of PASMCs under normoxia (21% O ₂ ); (C) is different degrees of hypoxia (10%, 5% and 1% O ₂ ) conditions The influence of PMVECs hypoxic condition culture medium containing HTSFCACE2 on the proliferation of PASMCs, *P<0.05vs.untransduced group, n=5; (D) Under the condition of 10% hypoxia, different concentrations of PMVECs containing HTSFCACE2 were lower Effect of oxygen-conditioned medium on the proliferation of PASMCs, *P<0.05vs.untransduced group, n=5.

Figure 8 is hypoxia promoting the progression of pulmonary hypertension in rats; where: (A) is the effect of 4 weeks of hypoxia on the mean left carotid arterial pressure (mCAP) of rats; (B) is the effect of 4 weeks of hypoxia The effect on right ventricular pressure (RVSP) in rats; (C) is the effect of 4 weeks of hypoxia on the right ventricular hypertrophy index [RV/(LV+S)] of rats; (D) is 4 weeks The effect of hypoxia on the area ratio (WA%) of rat pulmonary arterioles; (E) is the effect of hypoxia on the diameter ratio (WT%) of rat pulmonary arterioles in 4 weeks; (F) is the effect of 4 weeks low Effect of oxygen on lung tissue of rats; *P<0.05vs.Con group (normia), n=8.

Figure 9 shows that hypoxia reduces the expression of ACE2 and promotes the progression of pulmonary hypertension in rats; where: (A) and (B) are the impact and quantification of hypoxia on the expression of ACE2 protein in the lung tissue of rats; (C) is the effect of hypoxia on The influence of AngII content in rat lung tissue homogenate; (D) and (E) are hypoxia-reduced contents of ACE2 and Ang(1-7) in rat lung tissue homogenate; *P<0.05vs.Con group ( normoxia), n=8.

Figure 10 is the result of agarose gel electrophoresis of PCR products; lane M is marker, lane 1 is AAV-HTSFC (541bp), and lane 2 is AAV-HTSFCACE2 (2953bp).

Fig. 11 is the process of adeno-associated virus AAV-HTSFCACE2 improving hypoxic pulmonary hypertension in rats; wherein: (A) is the effect of AAV-HTSFCACE2 on the mean left common carotid artery pressure (mCAP) of rats; (B) is AAV- The effect of HTSFCACE2 on right ventricular pressure (RVSP) in rats; (C) the effect of AAV-HTSFCACE2 on the right heart hypertrophy index [RV/(LV+S)] in rats; (D) the effect of AAV-HTSFCACE2 on rat lung The effect of diameter ratio (WT%) of arterioles; (E) is the effect of AAV-HTSFCACE2 on the area ratio (WA%) of rat pulmonary arterioles; (F) is the effect of AAV-HTSFCACE2 on rat lung tissue Effect; *P<0.05vs.Normoxia group, #P<0.05vs.Hypoxia group, n=8.

Figure 12 shows the adeno-associated virus AAV-HTSFCACE2 up-regulates the expression of ACE2 in the lung tissue of hypoxic pulmonary arterial hypertension rats; wherein: (A) and (B) are the influence and quantitative results of AAV-HTSFCACE2 on the expression of ACE2 in the lung tissue of rats; (C) is the impact of AAV-HTSFCACE2 on the content of AngII in rat lung tissue homogenate; (D) is the impact of AAV-HTSFCACE2 on the content of Ang(1-7) in rat lung tissue homogenate; (E) The effect of AAV-HTSFCACE2 on the content of ACE2 in rat heart, liver, lung, kidney and other tissue homogenates; (F) is the impact of AAV-HTSFCACE2 on rat lung tissue detected by immunohistochemical staining; (G) is the immune Quantification results of histochemical staining; *P<0.05vs.Normoxia group, #P<0.05vs.Hypoxia group, n=8.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

1. Construction, identification and sequencing verification of recombinant plasmid HRE-Tie2-FC-ACE2

Whole gene synthesis 6×HRE sequence (rat HRE is: 5'GACTCCACAGTGCATACGTGGGCTTCCACAGGTCGTCTC3', see SEQ.ID.NO.1) and Tie gene (Tyrosine kinase with Id EGF homology domains) promoter (Mus musculus receptor tyrosine kinase Tie2gene, 5'-flanking region, access number AF022456.1, location: AF022456.1:1–223, referred to as Tie2 promoter). At the same time, synthesize signal peptide and hIgG1Fc fusion marker (SPFc: 5'ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCC3', see SEQ.ID.NO.2, signal peptide and fusion marker are fused in a sequence, which has two corresponding functions) and rat ACE2 coding sequence (see SEQ.ID.NO.5 for the specific sequence), and clone the above sequence into pUC57 (Shenzhen Baienwei Biotechnology Co., Ltd.) to obtain the template plasmid pUC57-HRE-Tie2-FC-ACE2; then use Amp ⁺ The pAAV-MCS vector plasmid (Shenzhen Baienwei Biotechnology Co., Ltd.) and the template plasmid pUC57-HRE-Tie2-FC-ACE2 were digested with MluI and BamHI, and the large fragment of the recovered pAAV-MCS vector plasmid was recombined with ACE2 The target gene (HRE-Tie2-FC-ACE2, where Tie2 represents the Tie2 promoter, FC represents SPFc) was ligated, transformed and shaken overnight to screen positive bacterial clones and extract bacterial plasmids. The expression of ACE2 in the recombinant target gene fragment is controlled by the HRE and Tie2 promoters. At the same time, the signal peptide and hIgG1Fc fusion marker are used to realize the secretion of ACE2 in PMVECs and cut off at the marker site. Therefore, the recombinant plasmid is used for the above-mentioned recombinant purpose. Gene HRE-Tie2-FC-ACE2 named (referred to as HT SFCACE2). A recombinant plasmid not containing the ACE2 coding sequence was used as a control plasmid, named HRE-Tie2-FC (HTSFC for short), and the expression vector structure is shown in FIG. 1 .

Both the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were identified by double enzyme digestion with MluI and BamHI, and the target gene bands of 2953bp and 541bp were respectively obtained by agarose gel electrophoresis (Figure 2 and Figure 3). The plasmid pAAV-MCS was digested with a large fragment and the recombinant target gene was recovered, and the sequence was verified. The result was exactly the same as the original sequence, and the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were successfully synthesized.

2. Cell experiments

1. Primary culture and identification of rat pulmonary microvascular endothelial cells (PMVECs) and rat pulmonary artery smooth muscle cells (PASMCs)

Primary PMVECs were cultured according to the modified tissue block method. The specific steps are: anesthetize and kill the rats (purchased from the Animal Center of the Fourth Military Medical University), quickly remove the lung tissue, take the lung tissue pieces at the outer edge of the lung in the ultra-clean workbench, and rinse the lung tissue with pre-cooled serum-free DMEM culture solution. Tissue pieces were cut several times, and cut into tissue pieces with a volume of about 1 mm ³ and evenly planted in culture bottles. Add about 2ml of DMEM culture solution containing 20% FBS, 90U/ml heparin sodium, 100U/ml penicillin and 100U/ml streptomycin, place the culture bottle vertically for 1 hour, then turn the bottle over, let the culture solution slowly submerge and cover the tissue Piece. After continuing to culture for 24 hours, the medium was changed, and the blood cells were sucked off. The morphology of ECs was observed, and the mixed cells were removed by differential digestion and differential adhesion. Subculture after the basic monolayer of cells at the bottom of the bottle is confluent. PMVECs were identified by immunohistochemical staining for factor VIII.

Primary PASMCs were cultured by tissue block adherence method. The specific steps are: anesthetize and kill the rats, quickly remove the lung tissue, quickly separate the main pulmonary artery and the secondary and tertiary internal pulmonary arteries in an ultra-clean workbench, rinse with pre-cooled serum-free DMEM culture medium, and peel off the adventitia , destroy the intima of the blood vessel, cut into 1mm ³ tissue pieces and evenly plant them in culture flasks. Add 2ml of DMEM culture solution containing 15% fetal bovine serum, place the culture bottle vertically for 2-4 hours, then turn over the bottle, let the culture solution slowly submerge and cover the tissue block. After continuing to culture for 24 hours, the medium was changed, and the growth status of PASMCs was observed under a microscope at the same time. Around 7-10d, elongated fusiform PASMCs could be observed crawling out from around the tissue block. When the PASMCs grew to 80% confluence, the PASMCs were passaged, and the expression of anti-α-SM-actin in the PASMCs was detected by immunohistochemical method to identify the PASMCs.

2. Effect of transient transfection of HTSFCACE2 and control plasmids on the expression of ACE2 in different types of cells and cell supernatants under normoxia (21% O ₂ ) conditions

Add 50 μl of the bacterial solution containing the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC to a sterile Erlenmeyer flask containing Amp ⁺ LB medium, and cultivate overnight at 250 rpm on a shaker at 37°C. The measured OD600 value is about 0.4-0.6. The plasmid was extracted according to the kit instructions, the OD value was measured, quantified, and stored at -20°C until use.

A549, 293 and NIH-3T3 cells (purchased from ATCC, USA) were revived and cultured with DMEM medium containing 10% FBS. 2-3 passages of PASMCs, PMVECs, A549, 293 and NIH-3T3 cells were used. Inoculate into 6-well plates at a concentration of 10 ⁵ /ml to continue culturing. Transfection was performed when the cells grew to 80%. Each cell was transfected with recombinant plasmid HTSFCACE2 and control plasmid HTSFC (specific process: 4 μg of plasmid, 8 μl of Lipofectamine ^TM 2000 transfection reagent and 5 ml of serum-free medium were fully mixed and added to a 6-well plate), and 5 Repeat hole. The transfection process was performed strictly according to the instructions of Lipofectamine ^TM 2000 transfection reagent from Invitrogen Company. After 12 hours of transfection, the cells and cell supernatant were collected respectively, and the proteins in the cells were extracted, and the expression of ACE2 in different types of cells was detected by Western blot method; the content of ACE2 in the supernatant of different types of cells was detected by ELISA method, strictly according to ELISA According to the instructions of the kit, the ACE2 concentration in the supernatant of the cells to be tested is calculated.

The results of the above experiments are: under normoxia (21% _O2 concentration) cells and cell supernatants of A549 cells, 293 cells, pulmonary artery microvascular endothelial cells (PMVECs) and pulmonary artery smooth muscle cells (PASMCs) contain a small amount of expressed ACE2 , while the expression of ACE2 in fibroblast (NIH-3T3) cells and cell supernatant was less (Fig. 4). After the above four kinds of cells were transfected with the recombinant plasmid HTSFCACE2 under normal oxygen conditions (21% _O2 concentration), the expression of ACE2 in the cells and in the cell supernatant did not increase significantly in A549, 293, NIH-3T3 and PASMCs cells, But it was significantly increased in PMVECs (Fig. 4). However, the transfection of the control plasmid had no significant effect on the expression of ACE2 in the cells of various types of cells and in the supernatant of cells. The experimental results showed that the recombinant plasmid HTSFCACE2 was highly specifically expressed only in the transfected PMVECs and had cell targeting.

3. After the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were respectively transfected into PMVECs, the content of ACE2 in the cell supernatant was detected after stimulation with different hypoxia (10%, 5% and 1% O ₂ )

Select the PMVECs cultured at passage 3-4 and inoculate them into 6-well plates at a concentration of 10 ⁵ /ml, and perform transfection after the cells grow to 80% (specific process: 4 μg of plasmid, 8 μl of Lipofectamine ^TM 2000 transfection reagent and 5 ml of serum-free culture base was mixed well and added to a 6-well plate), the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were transfected respectively, and 5 duplicate holes were made. Immediately after transfection, PMVECs were placed in a trigas culture incubator, stimulated with normoxia (21% O ₂ ) and different concentrations of hypoxia (10%, 5% and 1% O ₂ ), and continued to culture for 24 hours. The cells and cell supernatant were collected, the protein in the cells was extracted, and the expression of ACE2 in PMVECs was detected by Western blot method; the change of ACE2 content in the supernatant of PMVECs was detected by ELISA method.

The results of the above experiments showed that the expression of ACE2 in PMVECs cells gradually decreased with the decrease of oxygen concentration in the case of no plasmid transfection. After transfection of the recombinant plasmid HTSFCACE2, the expression of ACE2 in PMVECs cells was significantly increased (Figure 5A and B), and the increase rate of ACE2 expression increased with the decrease of oxygen concentration (Figure 5C).

In the case of no plasmid transfection, the content of ACE2 in the supernatant of PMVECs cells gradually decreased with the decrease of oxygen concentration. After transfection of the recombinant plasmid HTSFCACE2, the content of ACE2 in the supernatant of PMVECs cells increased significantly (Figure 6A), and the increase rate of ACE2 expression increased with the decrease of oxygen concentration (Figure 6B).

Transfection of the control plasmid had no significant effect on the expression of ACE2 in PMVECs cells and cell supernatant. Therefore, the recombinant plasmid HTSFCACE2 has hypoxia controllability at the cellular level.

4. Prepare PMVECs hypoxic condition culture solution, add it to PASMCs to observe the proliferation changes of PASMCs after stimulation with normoxia (21% O ₂ ) and different concentrations of hypoxia (10%, 5% and 1% O ₂ )

PMVECs were inoculated into 6-well plates at a concentration of 10 ⁵ /ml for culture, and transfected after growing to 80% (specific process: 4 μg of plasmid, 8 μl of Lipofectamine ^TM 2000 transfection reagent and 5 ml of serum-free medium were mixed thoroughly, and added to 6 wells plate), transfect the recombinant plasmid HTSFCACE2 and do 5 duplicate holes. Immediately after transfection, PMVECs were placed in a three-gas culture incubator, given 10% hypoxia stimulation, and after continuing to culture for 24 hours, the PMVECs supernatant was collected immediately, which was the PMVECs hypoxia-conditioned medium. PMVECs culture medium under hypoxic conditions is prepared immediately and cannot be frozen to avoid degradation of ACE2. After the control plasmid HTSFC was transfected into PMVECs, 10% hypoxia stimulation was also given, and the supernatant was collected immediately after continuing to culture for 24 hours, which was the control conditioned medium.

PASMCs in good growth state were taken, and 5×10 ³ cells/well were evenly inoculated in a 96-well plate, and DMEM culture solution containing 10% FBS was added. When the cells were 60% full, replace the serum-free DMEM medium and culture for 24 hours to synchronize them. The newly prepared PMVECs hypoxic condition culture fluid or control condition culture fluid containing the recombinant plasmid HTSFCACE2 was mixed with DMEM culture fluid containing 10% FBS in different proportions and added to the wells, and the PMVECs hypoxia condition culture fluid or control condition The mixing ratio of the culture medium is divided into 0%, 25%, 50% and 75%:

①0% group: Add 200 μL of DMEM culture solution containing 10% FBS + 0 μL of the PMVECs hypoxia-conditioned medium or control-conditioned medium to each well;

②25% PMVECs hypoxic conditioned medium group/control conditioned medium group: add 150 μL of DMEM medium containing 10% FBS + 50 μL of the PMVECs hypoxic conditioned medium or control conditioned medium to each well;

③50% PMVECs hypoxic conditioned medium group/control conditioned medium group: add 100 μL 10% FBS DMEM medium + 100 μL of the PMVECs hypoxic conditioned medium or control conditioned medium to each well;

④ 75% PMVECs hypoxia-conditioned medium group/control-conditioned medium group: add 50 μL of DMEM medium containing 10% FBS + 150 μL of the PMVECs low-oxygen-conditioned medium or control-conditioned medium to each well;

Then the PASMCs of the above groups containing or not containing the PMVECs hypoxic conditioned medium or the control conditioned medium were placed in different oxygen concentrations (21%, 10%, 5% and 1% O ₂ ), and each Oxygen concentration to do 5 replicate holes. Add MTT (5 mg/mL) and continue to incubate for 4 h, then add 150 μl of DMSO solution to each well, shake for 5 min, measure the absorbance OD value of each well at a wavelength of 490 nm on an automatic microplate reader, take 3 times of measurement results for each well, and calculate average value.

The results of the above experiments are: after culturing pulmonary artery smooth muscle cells (PASMCs) under normoxia (21% O ₂ ) and different concentrations of hypoxia stimulation (10%, 5% and 1% O ₂ ) for 48 hours, PASMCs The decrease of the concentration resulted in different degrees of proliferation, suggesting that hypoxia induces the proliferation of PASMCs (Fig. 7A). The recombinant plasmid HTSFCACE2 was transfected into PMVECs, and after 24 hours of hypoxia stimulation with 10% oxygen concentration, the collected PMVECs cell supernatant was PMVECs hypoxic conditioned medium (i.e. PMVECs hypoxic conditioned medium containing HTSFCACE2). The fresh PMVECs hypoxic condition culture solution was immediately added to PASMCs, and then continued to culture for 48 hours under normoxic (21%, O ₂ ) and different concentrations of hypoxic (10%, 5% and 1% O ₂ ) conditions Afterwards, the proliferation changes of PASMCs were detected. The PMVECs culture medium under hypoxic conditions containing HTSFCACE2 had no significant effect on the proliferation of PASMCs cultured under normoxic (21% O ₂ ) conditions ( FIG. 7B ). However, under different concentrations of hypoxic conditions (10%, 5% and 1% O ₂ ), PMVECs hypoxic conditioned medium containing HTSFCACE2 had a significant inhibitory effect on the proliferation of PASMCs induced by hypoxia (Fig. 7C), and its The inhibitory effect was dose-dependent, and the PMVECs hypoxic condition culture solution added according to the proportion of 50% could significantly inhibit the proliferation of PASMCs induced by hypoxia ( FIG. 7D ). The control plasmid HTSFC was transfected into PMVECs, and after 24 hours of hypoxia stimulation with 10% oxygen concentration, the collected supernatant of PMVECs cells was the control conditioned medium. The control conditioned medium had no effect on inhibiting the proliferation of PASMCs.

The above cell experiment results show that the recombinant plasmid HTSFCACE2 can only increase the expression of ACE2 in PMVECs according to the degree of hypoxia, and the PMVECs hypoxic condition culture medium containing HTSFCACE2 can reduce the proliferation of PASMCs in a dose-dependent manner, which has the target of cellular level. tropism, hypoxia controllability and effectiveness.

2. In vivo animal experiments

1. Copy the model of hypoxic pulmonary hypertension in rats, experimental grouping and detection of various indicators

SD rats were randomly divided into 5 groups, 8 in each group:

①Normoxia group: Rats were placed in a normoxia environment for 28 days.

②Hypoxia 7 days group (Hypoxia 7d): Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours a day for 7 consecutive days.

③ Hypoxia 14 days group (Hypoxia 14d): Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours a day for 14 consecutive days.

④ Hypoxia 21 days group (Hypoxia 21d): Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours a day for 21 days in a row.

⑤ Hypoxia 28 days group (Hypoxia 28d): Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours a day for 28 consecutive days.

The rats in the normoxia group were placed in the animal room and raised under natural conditions (atmospheric pressure in Xi’an area was about 718mm Hg, pO2 was _150.6mmHg , and the oxygen concentration was about 21%); the rats in the hypoxia group were placed in a hypobaric hypoxic cabin (the pressure in the cabin is 380mm Hg, pO2 is reduced to _79.6mmHg , which is equivalent to the oxygen content at an altitude of 5540 meters, and the oxygen concentration is reduced by about 10%) 8 hours a day for 28 consecutive days; soda lime and desiccant are used in the low-pressure hypoxic cabin Deodorize and absorb CO ₂ . After 28 days, the relevant indicators of pulmonary hypertension in rats in each group were detected.

Detection of hemodynamic indicators: anesthetize and fix the rats, make an incision in the middle of the neck, separate and ligate the distal ends of the left common carotid artery and the right external jugular vein, and clamp the proximal ends of the blood vessels. A small opening was cut with ophthalmic scissors, and polyethylene catheters filled with 0.5% heparin solution were inserted into the left common carotid artery and the right external jugular vein respectively. One end of the catheter was left in the blood vessel and tied and fixed. The other end was connected with a pressure transducer to record the mean left common carotid arterial pressure (mCAP) and peak right ventricular systolic pressure (RVSP) of the rat.

Measurement of right ventricular hypertrophy index RV/(LV+S)×100%: Cut open the rat sternum to expose the heart; remove the tissues and blood vessels around the heart, as well as the left and right atrium, atrial appendage, etc., find the conus pulmonary artery, and follow the pulmonary artery Cones down the right ventricle (RV) and weighed. The remaining tissue was also weighed as the weight of the left ventricle and septum (LV+S). Calculate the right ventricular hypertrophy index [RV/(LV+S)×100%] according to the measured value to reflect the degree of right ventricular hypertrophy.

Lung tissue paraffin section preparation and HE staining: along the pulmonary hilum, cut the tissue block of the right upper lobe of the rat about 1cm × 2cm, put it in the embedding frame, and put 10% neutral formaldehyde together with the embedding frame After fixing in the buffer for 24h, the embedding frame was taken out and placed in 70% ethanol solution. Then dehydrated, embedded, and made into paraffin blocks. Paraffin block sections were dewaxed to water for HE staining to detect changes in pulmonary arterioles.

Image analysis of lung tissue slices: observe HE stained slices under a microscope, select pulmonary arterioles with an outer diameter of less than 50-100 μm, use image analysis software to collect and analyze blood vessel images, and measure the inner, outer diameter, and wall thickness of blood vessels and blood vessel area, and then calculate two indicators reflecting the thickening of the blood vessel wall according to the measured values, namely WT% (tube wall thickness/outer diameter×100%) and WA% (tube wall area/total blood vessel area×100%) .

ELISA method to detect the content of ACE2, AngII and Ang(1-7) in lung tissue homogenate: cut a small piece of lung tissue at the same location, accurately weigh 100mg tissue, put it into EP tube, and add 1ml normal saline , and grind with a hand-held homogenizer to prepare a tissue homogenate. Then the EP tube was centrifuged at 4500 rpm for 10 min at 4°C, and the supernatant was aspirated. According to the instructions of the ELISA kit, the contents of ACE2, AngII and Ang(1-7) in the lung tissue homogenate were detected.

Detect the expression of ACE2 in lung tissue by Western blot: Cut out a small piece of lung tissue at the same site, accurately weigh 100 mg of tissue, and add lysis solution RIPA at a ratio of 10 mg/100 ul of tissue/lysate. Cut the tissue as quickly as possible with small ophthalmic scissors, homogenize it fully, and lyse it on ice for 30 minutes. Centrifuge the specimen in a centrifuge at 12000 rpm for 5 min at 4°C and collect the supernatant. The supernatant is the cell protein. Protein content was quantified by the Coomassie brilliant blue method. Add the prepared protein standards and protein samples into the 24-well plate with a pipette, measure the OD value of the samples at a wavelength of 595nm, combine the concentration of the protein standard, and compile the protein concentration standard curve. Calculate the corresponding protein concentration according to the curve and the absorbance of the protein sample, and determine the loading volume of the protein sample. According to the protein sample concentration, add different deionized water and 5×SDS loading buffer, place in a 95°C water bath and cook for 10 minutes to promote protein denaturation. According to the molecular weight of ACE2, 12% of the lower glue and 6% of the upper glue were prepared respectively. Then electrophoresis is carried out, and the electrophoresis buffer is added to the electrophoresis tank. Protein samples were centrifuged at 12,000 rpm for 1 min before use. Add protein markers and protein samples to the sample wells with a pipette, and the sample volume is about 50 μg protein samples per well. Turn on the power and carry out SDS-PAGE electrophoresis. The upper glue uses 80V voltage, while the lower glue adjusts the voltage to 120V. Turn off the power when the bromophenol blue reaches the bottom of the gel. Next, transfer the membrane, take out the gel, cut off the upper layer of gel, and soak the lower layer of gel in the transfer buffer. Assemble the transfer clip, unfold the clip in the transfer solution, place the liner, three layers of filter paper, gel, NC membrane, three layers of filter paper and the liner in turn on the black surface, roll gently with a glass rod to drive away each Air bubbles between layers, close clamps, and place in transfer tank. Turn on the power supply and transfer for about 2h with a voltage of 100V. After transfer, soak the NC membrane in Ponceau staining solution. Mark the position of the protein marker, and cut the membrane according to the molecular weight. Put the marked Marker and the trimmed membrane into a plate containing TBST buffer, shake it at 80rpm for 10min, and wash away Ponceau. The cut NC membrane was blocked with 10% skimmed milk powder at 37°C for 1 h. Then the cut NC membrane was added with ACE2 primary antibody (1:200 dilution) or β-actin primary antibody (1:10000 dilution), incubated on a shaker at 80rpm for 2h, and shaken overnight at 4°C for antibody hybridization incubation. Next, put the NC membrane into a plate containing TBST buffer, wash the membrane 3 times×10min. Add goat anti-mouse IgG secondary antibody (1:5000 dilution) and place on a shaker at 80rpm for 1h incubation. Then the NC membrane was rinsed three times with TBST buffer, about 10 min each time. Add ECL chemiluminescent reagent to the NC membrane, develop it on a chemiluminescent instrument, and save the picture. The ACE2 protein is 92kD, while the β-actin protein is 43kD. The ratio of the gray value of their bands is the relative expression of ACE2, that is, the gray value of the ACE2 band/the gray value of β-actin.

The results of the above experiments are as follows: the rat model of hypoxic pulmonary hypertension was replicated for 4 weeks. During this process, with the prolongation of hypoxic time, the peak systolic pressure of the right ventricle (RVSP) of the rats gradually increased (Fig. 8B), reflecting The right heart hypertrophy index [RV/(LV+S)]% gradually increased (Fig. 8C), and hypoxia caused the rat pulmonary arteriole smooth muscle layer to gradually thicken, and the lumen narrowed (Fig. 8F), representing the pulmonary arteriole The thickening indicators WA% and WT% both increased significantly (Fig. 8D, E). And the expression of ACE2 in hypoxia-induced lung tissue decreased significantly with time (Figure 9A and B), and the content of AngII in lung tissue homogenate increased significantly (Figure 9C), while the contents of ACE2 and Ang(1-7) significantly decreased decreased (Figure 9D and E).

The results of the above in vivo animal experiments showed that hypoxia leads to the disorder of the RAS system in the lung, the expression of ACE2 is significantly reduced, and the expression of AngII is relatively hyperactive, which in turn induces the development of hypoxic pulmonary hypertension.

2. Adeno-associated virus AAV-HTSFCACE2 and AAV-HTSFC construction, collection and virus titer determination

Resuscitate frozen 293AAV cells (Shenzhen Baienwei Biotechnology Co., Ltd.), culture them with ¹⁰ % FBS DMEM culture medium, inoculate them in a 10cm culture dish, adjust the number of 293AAV cells to about 1-5×106, and continue to cultivate Keep cells up to 80% confluent. The medium was changed 2 hours before transfection, and then the plasmid DNA was transfected into 293AAV cells according to the instructions of the HET transfection kit from Shenzhen Baienwei Company. The transfected 293AAV cells were cultured for 4-6 hours before changing the medium. After 12-18 hours of transfection, use a pipette to suck off the culture solution in the culture dish, and add 10 ml of fresh complete culture solution containing 1% double antibody to continue culturing. After culturing for 48 hours, the cell mixture was collected and placed in a centrifuge tube, placed in a water bath at -80°C and 37°C, repeatedly frozen and thawed three times, centrifuged at 3000 rpm for 10 minutes, the supernatant was collected, concentrated and purified, and stored at -80°C.

Design Real time PCR primers:

hGHpolyA Forward: 5'-CAAGCGATTCTCCTGCCTCA-3' (see SEQ.ID.NO.4)

hGHpolyA Reverse: 5'-ACGCCTGTAATCCCAGCAAT-3' (see SEQ.ID.NO.3)

According to the requirements of the experiment, take 20ul of the concentrated virus solution of AAV-HTSFCACE2 and AAV-HTSFC to be tested, and make 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , and 10 ⁷ dilutions respectively, and then construct a Realtime PCR reaction system for PCR reaction. Based on the measured Ct values, the copy numbers in the AAV samples were calculated.

3. Identification of adeno-associated virus AAV-HTSFCACE2/AAV-HTSFC

Extraction of viral genomic DNA: 293 cells were cultured to more than 80% confluency, and 200 μl of AAV-HTSFCACE2 or AAV-HTSFC virus cryopreservation solutions were added to continue culturing for 36 hours. When the cells are round but not yet floating, pipette off the medium and rinse the cells once with PBS. Add 800 μl of cell lysate (containing 0.6% SDS, 10 mM EDTA and 100 μg/ml proteinase K) to each bottle, incubate at 56°C for 1 hour, then add 200 μl of 5M NaCl to each bottle, mix well, and place on ice for 1 hour , and then the collected cells were centrifuged at 12000 rpm for 10 min at 4°C. Aspirate the supernatant, add an equal volume of phenol, chloroform and isoamyl alcohol mixture (mixed according to the ratio of 25:24:1), centrifuge at 12000rpm for 15min, collect the supernatant, and add 1/9 volume of 3M NaAc and 2 times volume of absolute ethanol and placed at -20°C for 30 min. Then centrifuge at 12000rpm for 15min, discard the supernatant, and vacuum dry the water. Then add 1ml of 70% ethanol to resuspend, centrifuge at 10000rpm for 10min, and discard the supernatant. Place the EP tube at room temperature for 10 minutes to dry the water, then add 40ul of deionized water to dissolve the extracted DNA, and store it at -20°C.

PCR identification: construct a PCR reaction system, carry out PCR reaction, take 5mL PCR products and carry out 1% agarose gel electrophoresis respectively, and check the PCR results.

According to the above experiments, the recombinant plasmid HTSFCACE2 and the control plasmid HTSFC were respectively transfected into 293AAV cells, the cell mixture was collected, centrifuged, packaged into adeno-associated virus, and the virus titer was measured. Virus genomic DNA was extracted for Real-time PCR, and the PCR products were subjected to 1% agarose gel electrophoresis for virus identification. The PCR product identification results are shown in FIG. 10 , and the adeno-associated virus titers are shown in Table 1. The results showed that the virus was packaged successfully, the virus titer was good, and it could be used in animals.

Table 1. Titers of AAV virus

4. Adeno-associated virus AAV-HTSFCACE2 improves the process of hypoxic pulmonary hypertension in rats

SD rats were randomly divided into 4 groups, 8 in each group:

①Normoxia group: Rats were placed in a normoxia environment for 28 days.

② Hypoxia group (Hypoxia): Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours a day for 28 days in a row to replicate the model of hypoxic pulmonary hypertension in rats.

③Hypoxia+AAV-HTSFCACE2 group: Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours every day, and adeno-associated virus AAV-HTSFCACE2 was instilled into the nose at one time on the 15th day, and the hypoxia continued until 28 days .

④Hypoxia+AAV-HTSFC group: Rats were placed in a hypobaric hypoxic chamber with an oxygen concentration of 10% for 8 hours every day, and adeno-associated virus AAV-HTSFC was instilled into the nose at one time on the 15th day, and the hypobaric hypoxia continued until 28 days .

When rats are instilled nasally, the supine position is fixed after anesthesia, the head is raised, and a specific amount of recombinant adeno-associated virus is sucked with a pipette, and slowly dripped into the nostril of the rat. inhale. The amount of virus in each rat was about 3×10 ¹¹ μg/ml. After intranasal instillation, the rats woke up naturally, and the status of the rats was observed. Those in good condition continued the hypoxic treatment.

Hypoxic measures are the same as mentioned above. After 28 days, the relevant indicators of pulmonary hypertension in rats in each group were detected, the same as above. The expression of ACE2 in lung tissue was detected by Western blot, the contents of ACE2, AngII and Ang (1-7) in lung tissue homogenate were detected by ELISA, and the expression of ACE2 in pulmonary arterioles was examined by immunohistochemistry.

Detect the expression of ACE2 in rat pulmonary arterioles by immunohistochemical method: after dewaxing the prepared rat lung tissue paraffin sections to water, follow the steps below:

(1) Wash with xylene for 10min×4;

(2) Wash with 100% ethanol for 10min×2;

(3) Wash with 95% ethanol for 5 minutes;

(4) Wash with 90% ethanol for 5 minutes;

(5) Rinse with tap water for 5 minutes;

(6) Incubate with 3% H ₂ O ₂ deionized water at room temperature for 30 min;

(7) Wash with distilled water for 5min×3;

(8) Immerse the slices in 0.01M citrate buffer, place in a microwave oven for antigen retrieval, heat to boiling at high heat, then maintain at medium heat for 10 minutes, take it out and cool naturally at room temperature;

(9) Add normal rabbit serum working solution for sealing dropwise, room temperature for 30 minutes, pour off excess liquid, do not wash;

(10) Add 1:50 dilution of rabbit anti-rat ACE2 monoclonal antibody (1:200) dropwise, overnight at 4°C in a humid chamber, wash with PBS for 5min×3;

(11) Take out the wet box and rewarm at 37°C for 1 hour;

(12) Add 1:50 diluted peroxidase-labeled goat anti-rabbit IgG dropwise, wash at 37°C for 30 min, and wash with PBS for 5 min×3;

(13) DAB color development, fully rinsed with tap water;

(14) counterstained with hematoxylin, dehydrated, transparent, and mounted

(15) Observed under a light microscope, it can be seen that the cell membrane is yellowish brown, which is ACE2 positive cells. Under the 400X field of view, three pulmonary arterioles with a diameter of 200-500 μm were selected, and the average integrated optical density (IOD) of the positive particles in the smooth muscle layer of the pulmonary arterioles was measured and analyzed with the IMAGE-PRO plus6 microscopic image analysis system for analysis and processing.

The results of the above experiments are as follows: After replicating the 4-week hypoxic pulmonary hypertension rat model, and administering the adeno-associated virus AAV-HTSFCACE2 or the control virus AAV-HTSFC to the rats once in the second week, in the form of nasal drops, Hypoxia was then continued until week 4. The results showed that adeno-associated virus AAV-HTSFCACE2 could significantly reduce RVSP, [RV/(LV+S)]%, WA% and WT% in rats (Fig. 11B, C, D, E), and pulmonary small vessel morphogenesis Significant changes, slightly thickened arteriole muscularis, lumen narrowing is not obvious (Figure 11F). This part of the experiment fully demonstrates that the adeno-associated virus AAV-HTSFCACE2 has a significant therapeutic effect on rats with hypoxic pulmonary hypertension, reducing right ventricular pressure, reducing right ventricular hypertrophy, inhibiting the structural changes of small pulmonary vessels, and adeno-associated virus AAV-HTSFCACE2 had no significant effect on the mean left common carotid artery pressure (mCAP) in rats (Fig. 11A). Rats given the control virus AAV-HTSFC did not have the above symptoms.

In addition, adeno-associated virus AAV-HTSFCACE2 can up-regulate the expression of ACE2 in lung tissue (Figure 12A and B), reduce the content of AngII in lung tissue homogenate (Figure 12C), and increase the content of Ang(1-7) (Figure 12D) . The results of immunohistochemical staining showed that after administration of adeno-associated virus AAV-HTSFCACE2, the expression of ACE2 in the endothelial tissue of pulmonary arterioles increased (Fig. 12F and G). And the content of ACE2 in rat heart, liver, lung, kidney and other tissues was detected by ELISA method, and it was found that after administration of adeno-associated virus AAV-HTSFCACE2, the content of ACE2 was only significantly increased in lung tissue, and the change in other tissues was not obvious ( FIG. 12E ), suggesting that after the adeno-associated virus AAV-HTSFCACE2 enters the rat body, the expression of ACE2 is only increased in the pulmonary arteriole endothelial tissue, which has tissue specificity and effectiveness. Rats given the control virus AAV-HTSFC did not have the above symptoms.

The results of the above in vivo animal experiments showed that adeno-associated virus AAV-HTSFCACE2 improved the progression of hypoxic pulmonary hypertension at the animal level.

In a word, the present invention utilizes gene recombination technology, combined with the characteristics of ACE2 to finely regulate the renin-angiotensin (RAS) system in the lung, the expression of Tie2 gene has the specificity of vascular endothelial cells, and the hypoxia response element (HRE) is regulated by HIF. -1α regulation (increased expression of hypoxia-inducible factor-1α (HIF-1α) during hypoxia is associated with decreased oxygen partial pressure, and HIF-1α expression only increases in pulmonary vessels during hypoxia, while Less increase in systemic circulation) and other characteristics, construction of ACE2 expression vector driven by HRE-enhanced Tie2 promoter, so that ACE2 is highly expressed in hypoxic pulmonary artery endothelial cells, and then regulates pulmonary artery smooth muscle cells through paracrine action to achieve hypoxia controllable Inhibit the contraction of the pulmonary artery and reverse the structural reconstruction of the pulmonary artery in a targeted and targeted manner, providing new means and strategies for the prevention and treatment of hypoxic pulmonary hypertension, and also for diabetic gangrene, deep vein thrombosis, ischemic heart disease, etc. Or/and provide ideas and strategies for the prevention and treatment of a series of diseases related to ischemia.

<110> The Fourth Military Medical University of the Chinese People's Liberation Army

<120> Controllable up-regulation of ACE2 expression vector for targeted prevention and treatment of hypoxic pulmonary hypertension

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Claims (7)

1. An ACE2 recombinant expression vector, which is characterized in that: comprises a hypoxia response element, a promoter, a signal peptide and an ACE 2gene, wherein the hypoxia response element is selected from HRE, and the promoter has specificity to vascular endothelial cells;

the expression vector specifically comprises a 6 × HRE gene promoter, a Tie2gene promoter, a signal peptide, an hIgG1Fc fusion marking element and an ACE 2gene which are sequentially arranged.

2. A method of constructing the expression vector of claim 1, wherein: the method comprises the following steps:

synthesizing HRE, a promoter, a signal peptide and an ACE2 gene; the individual elements synthesized were cloned into an expression vector backbone.

3. Use of the expression vector of claim 1 for the preparation of a medicament for the prevention and/or treatment of hypoxic pulmonary hypertension and complications thereof.

4. Use according to claim 3, characterized in that: the expression vector has cell targeting property, hypoxia controllability and effectiveness of inhibiting proliferation of pulmonary artery smooth muscle cells, wherein the targeting property shows that ACE2 is specifically and highly expressed in pulmonary microvascular endothelial cells after the expression vector is transfected, the controllability shows that the increase rate of the expression of the ACE2 is increased along with the reduction of oxygen concentration, and the effectiveness shows that the expression vector has an effect of inhibiting the proliferation of the pulmonary artery smooth muscle cells induced by hypoxia.

5. Use according to claim 3, characterized in that: the drug is selected from a lung microvascular endothelial cell culture solution containing the expression vector or adeno-associated virus packaged with the expression vector.

6. Use according to claim 3, characterized in that: the medicine adopts a nasal administration mode.

7. Use of the expression vector of claim 1 for the preparation of a medicament for the prevention and/or treatment of hypoxia and/or ischemia-related diseases, characterized in that: the disease is selected from one or more of diabetic gangrene, deep vein thrombosis and ischemic heart disease.

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