CN102533653B - 5-HT nerve cells induced by MSCs and ASCs and microencapsulation preparation method and application thereof - Google Patents

Abstract

5-HT nerve cells induced by MSCs and ASCs, including the following steps: (1) MSCs or ASSCs culture and passage: ① primary culture; ② digestion and passage; (2) precursor induction; (3) function induction; (4) The performance is stable and cultivated. The microencapsulation preparation method of 5-HT nerve cells induced by MSCs and ASCs adopts the ANano-P-ANano (sodium alginate + nanoparticle-polyamic acid-sodium alginate + nanoparticle) method, through an electrostatic microcapsule generator Microencapsulate the 5-HT nerve cells induced by MSCs and ASCs Microencapsulation; each microcapsule contains 20 or 30 5-HT nerve cell groups, so that the cells in the microcapsules can fully contact the microcapsule wall, easy to attach and nourish Exchange, that is, to make microencapsulated inducible 5-HT nerve cells. The application of the microencapsulated inducible 5-HT nerve cells is used for preparing medicines for treating depression or anticancer medicines. The microencapsulated inducible 5-HT cells of the present invention, the microencapsulated human 5-HT nerve cell central transplantation therapy, will provide a new approach for clinical treatment of depression and anticancer treatment.

Description

5-HT nerve cells induced by MSCs and ASCs and its microencapsulation preparation method and application

technical field

The invention belongs to the field of biomedicine, and relates to stem cell-induced 5-HT nerve cells and cell isolation (microencapsulation) technology and method, specifically, MSCs, ASCs-induced 5-HT nerve cells and a microencapsulation preparation method and method thereof. application. the

Background technique

(1) 5-HT nerve cells have a wide range of central connections and regulation

5-hydroxytryptamine (5-hydroxytryptamine, 5-HT) nerve cells or neurons are mainly located in the raphe nucleus of the brainstem, the compact part of the substantia nigra, the caudal locus coeruleus and the hypothalamus, and its outgoing fibers project almost to the entire central nervous system, ascending to the edge Systemic (hippocampus, septum, hypothalamus) and neocortex (frontal, parietal, occipital, temporal, insular) broad areas; anterior horn somatic motor and lateral horn visceral motor (sympathetic and parasympathetic) neurons descending to spinal cord . Its physiological function shows that 5-HT neurons and fibers in raphe nucleus, solitary tract nucleus, hypothalamus, amygdala, hippocampus, etc. are related to sleep regulation, and neurons in hippocampus, amygdala, and frontal lobe coordinate emotions and behaviors. We used 2.5% paraformaldehyde plantar injection of animal model experiments, through the dorsal hippocampal stimulation analgesia showed that the expression of 5-HT in the central canal gray matter (PAG) of the midbrain and the dorsal horn of the spinal cord increased, indicating that 5-HT and its neurons Participate in pain relief. Some experiments also show that the head of the raphe nucleus is destroyed, and non-rapid eye movement sleep disappears completely. The use of 5-HT synthesis inhibitors can cause complete insomnia and induce abnormal discharge of brain waves. Injecting 5-HT acid into the lateral ventricle can eliminate this phenomenon. These experimental results confirm that 5-HT neurons and their transmitter 5-HT participate in the multifunctional regulation of sleep, emotion, behavior and analgesia, that is, 5-HT neurons play an important role in regulating the normal function of the central nervous system. the

(2) Long-term stress leads to decompensation of 5-HT nerve cell function-expressing depression

Research data show that 5-HT neurons in the central system release 5-HT to participate in stress regulation, and when the expression level of 5-HT in the central nervous system decreases, psychological stress injury is prone to occur. This kind of psychological stress injury can cause two consequences: ① Increase the risk of depression and the severity of depression. After the stimulation of sexual event → the autonomic nervous system is overexcited → through a series of reactions, the levels of norepinephrine and adrenaline increase, and at this time, the release of these two hormones is due to the low level of 5-HT released by the central 5-HT neurons Feedback regulation can not be normal, resulting in persistent hyperfunction of the adrenal axis; ② low central 5-HT level → insufficient regulation of the hippocampus, since the hippocampus is an important part of the central limbic system, the 5-HT → hippocampal pathway is not clear The reason is that the noxious stress that starts prematurely has a blocking effect, so the hippocampal dysregulation caused by the reduction of 5-HT level can cause behavioral "disinhibition" (oversensitivity to environmental stimuli), and it is prone to stress-induced body injury. Many research materials have also proved that the abnormality of neurotransmitters in patients with depression is mainly manifested in the decrease of 5-HT concentration in blood and cerebrospinal fluid, and autopsy also found that the content of 5-HT in brain tissue decreased. In addition, some studies on patients with depression in the Vietnam War also suggested that the decrease of 5-HT content in the body was closely related to the loss of normal self-control ability during the war and even the "post-war syndrome" after the war. Other studies have shown that 5-HT has an inhibitory effect on the high-level brain regions excited by NE neurons; and highly excited NE neurons can also inhibit the activity of 5-HT neurons in the raphe nucleus. When 5-HT decreases, the NE pathway will continue to be excited, causing insomnia, anxiety, depression, etc., which will lead to another decrease in 5-HT, which will worsen sleep and mood disorders. The two cause and effect each other, thus forming a vicious circle. Therefore, based on the reduction of 5-HT in the brain of depression, or the depletion of 5-HT or the low level of its synthesis and release as the cause of the disease, 5-HT reuptake inhibitors are clinically used to treat depression, and some patients have achieved better results. Effect. However, some patients are ineffective, and even have serious side effects, which are likely to be refractory or severe depression, due to the low level of central 5-HT. We use an internationally recognized animal model of chronic stress, and the transplantation of 5-HT neurons in the subarachnoid space can prevent the occurrence of animal behavior abnormalities and multiple organ damage, indicating that 5-HT cell transplantation can prevent and treat depression or stress Injury to the body. the

(3) Low central 5-HT level leads to peripheral organ lesions - susceptibility to canceration

Studies on the mechanism of tumorigenesis have reported that the low level of central 5-HT may make some organ cells susceptible to cancer or cancer cells are prone to deterioration (evolution), and the mechanism can be adjusted through the center→peripheral (vagus, etc.) nerves→organ cells. Low and neuroendocrine adrenal axis hyperfunction, excessive secretion of glucocorticoids, epinephrine and norepinephrine, resulting in low energy metabolism of organ cells and toxic molecules - excitatory amino acids and oxygen free radicals (inducible nitric oxide iNO, Accumulation of malondialdehyde (MDA) → caused by organ cell resistance to damage or repair dysfunction, such as esophageal squamous cell carcinoma (ESCC), although etiology has already suggested that esophageal cancer is related to the environment (food mildew, nitrosamines), genetics However, studies in recent years have shown that esophageal cancer is a chronic disease, psychological stress factors (trauma or depression) play an important role in esophageal carcinogenesis, and the neuro-cholinergic anti-inflammatory pathway (cholinergic antiinflammatory pathway) , CAP), the protective effect of the vagus nerve on visceral damage caused by stress in the CAP pathway also requires the activation of 5-HT function in the raphe nucleus of the brainstem. Because the central 5-HT is involved in stress regulation, chronic psychological stress or depression can lead to a long-term low level of 5-HT in the brain. Therefore, psychological stress → low central 5-HT level is closely related to the carcinogenesis of esophageal mucosal epithelial cells. In addition, Toh S et al. investigated and compared the use of antidepressant 5-HT reuptake inhibitors, and found that the incidence of lung cancer among the drug users was reduced. Pyroja S induced liver tumors through hepatectomy + N-nitrosodiethylamine (NDEA), and found that the binding force of 5-HT2C receptors in the brainstem and cerebral cortex increased during the formation of liver tumors, which indicated that the level of 5-HT was too high. Low. El-Salhy et al. combined octreotide (a hemostatic drug in the gastrointestinal tract), galanin (neurotransmitter regulating peptide - can regulate appetite, induce apoptosis, etc.) and 5-HT to have better anticancer effects on gastrointestinal cancer cells . Our recent animal experiments using 5-HT cell subarachnoid transplantation to prevent and treat stress + nitrosamine esophageal cancer showed that chronic stress can lead to accelerated and aggravated esophageal carcinogenesis, and 5-HT cell transplantation can slow down or reduce esophageal carcinogenesis in mice. Reduced incidence. the

(4) Neural pathway dysfunction, one of the mechanisms of central 5-HT deficiency and peripheral organ lesions

Studies on the relationship between central 5-HT neuron → vagus nerve cholinergic pathway abnormal regulation and visceral injury or cell canceration have shown that the pathway from central 5-HT neuron → vagus nerve cholinergic → visceral pathway has been clarified, through the vagus nerve and its Postganglionic fibers release ACh to act on the cell's AChR (glycoprotein embedded in the cell membrane) to regulate internal organs or effector cells. Grozio et al. reported that choline receptors can regulate cell function, proliferation, transformation and differentiation, and the subtype difference of receptors plays an important role in cell proliferation-differentiation or tumorigenesis-malignant transformation. Using nAChR-α7 or mAChR-3 antagonists can inhibit the proliferation of lung cancer cells. Studies have confirmed that the lower the degree of differentiation of cancer cells, the more significant the reduction of AChRs subtypes, and the more binding sites are lost. We cultured highly and poorly differentiated esophageal cancer cells (highly differentiated EC9706 and poorly differentiated EC109) with ACh for 21 days respectively, and found that ACh could make esophageal cancer cells AChR, gene damage repair factor Prx1, cell differentiation calcium-dependent regulatory protein Annexin2 and cytoskeleton The expression of protein Keratins was up-regulated, and the morphology tended to be differentiated. the

Our recent animal experiments showed that severing the vagus nerve can accelerate the carcinogenesis induced by nitrosamines on the esophageal epithelium, which may be related to weakening the protective effect of the CAP pathway. Combining TPH2 gene polymorphism as redox, participating in sexual susceptibility and weakened vagus nerve function can accelerate stress + nitrosamine-induced esophageal carcinogenesis. It is characteristic that some patients with esophageal cancer will heal without treatment or survive for many years, and the degree of lesion remains unchanged. This suggests that human cancer cells have the ability to induce differentiation (reversible), and through the benign regulation of central-vagus nerve-organ function, it can inhibit For cancer cells, control the disease or achieve the purpose of curing, that is, the improvement of the central 5-HT system → vagus nerve pathway and postganglionic neurotransmitter AChR function has a benign impact on the transformation and differentiation of related cancer cells. This shows that the central 5-HT and the vagus nerve play an important role in inhibiting the carcinogenesis and anti-cancer of the relevant internal organ cells. the

(4) Central 5-HT nerve cell decompensation - derived from acquired and congenital factors

Studies have suggested that there are two reasons for the low level of central 5-HT: acquired and congenital: ① The reason for the acquired deficiency is that 5-HT neurons in the raphe nucleus of the brainstem send fibers to the limbic system, cerebral neocortex, and spinal cord. Extensive projections to the visceral motor nucleus (sympathetic and parasympathetic) are the basis of the 5-HT stress-regulating pathway. The 5-HT pathway in the center can regulate the two systems of NE and ACh, and NE can lead to the reduction and exhaustion of 5-HT neurons. In patients with chronic stress and depression, the content of 5-HT in blood, cerebrospinal fluid (CSF), and brain tissue decreased, and the content of total 5-HT receptors in the hippocampus decreased. After treatment, the receptor density can be restored, while blocking 5-HT2A Both /2C receptors can cause anxiety and insomnia, and aggravate 5-HT depletion caused by NE excitation. It can be seen that the continuous hypertonic state of the NE system initiated by chronic stress can lead to the depletion of central 5-HT, and the depletion of central 5-HT can progressively aggravate the excitement and emotional disturbance of the NE system. A vicious circle, which leads to the progressive depletion of central 5-HT. ②The reason for the low congenital 5-HT level is the congenital small hippocampus of some patients. This kind of congenital small hippocampus may originate from the rate-limiting enzyme of central 5-HT neuron synthesis of 5-HT found in the latest research-tryptophan Hydroxylase (Tryptophan hydroxylase, TPH) gene polymorphism. TPH1 mainly plays a role in the peripheral system, while TPH2 plays a role in the central; the mouse C1473G mutation causes proline at position 447 to be replaced by arginine, and the level of central 5-HT can be reduced by 30% to 70%, and it appears to be related to central 5-HT. - Behavioral changes associated with dysfunction of the HT system. When TPH2 carrying the G allele was expressed in PC12 cells, the synthesized 5-HT was only 45% of wild-type A. This suggests that the low level of central 5-HT is caused by the abnormal function of the primary neuron gene and the combination of predisposing factors, which is also an important basis for some people to be prone to depression or their organ cells to be prone to cancer. the

(5) Central 5-HT cell transplantation improves central 5-HT decompensation - broad application prospects

So far, the clinically used antidepressant and anticancer drugs bring relatively large toxic and side effects. In summary, if 5-HT cell center transplantation is given to patients in long-term chronic stress state, depression, and related cancer patients, the 5-HT released by it can compensate for the effect of regulating the center or anti-cancer. Moreover, this method can be used to prevent certain special events or special situations such as major earthquakes, wars, etc., and the self-out-of-control state or emotional abnormality syndrome caused by psychological stimulation or pressure can also play a preventive effect. At present, the prevention and treatment of depression or cancer in foreign countries is also paying more and more attention to the comprehensive treatment experiment of joint nervous system-transmitter regulation, including its agonists and antagonists, which has shown good prospects. the

(6) Clinical application of 5-HT nerve cell source-stem cell induced functional cells and microencapsulation

The sources of adult 5-HT nerve cells that can be used for therapeutic transplantation are extremely scarce, and allografts have rejection reactions, so it is difficult to promote their application in clinical transplantation. At present, stem cells have become an ideal solution for cell or organ replacement therapy, and it has become an inevitable trend to use autologous stem cells or allogeneic histocompatibility-inducing methods to obtain 5-HT nerve cells that can be used in human transplantation. Existing studies have shown that human bone marrow and fat are rich stem cell pools, easy to obtain and safe, and the bone marrow mesenchymal stem cells MSCs and ASCs can differentiate into multifunctional. However, there must be corresponding methods and conditions to drive stem cells to differentiate into required functional cells, and no mature technology and methods have been found to promote the differentiation of stem cells into 5-HT nerve cells at home and abroad. Due to the transplantation of mature cells induced by autologous stem cells, mild rejection may sometimes occur, and an effective way to avoid rejection is to isolate the transplanted cells from the immune system-microencapsulation. the

Contents of the invention

The object of the present invention is to provide 5-HT nerve cells induced by MSCs and ASCs. the

The object of the present invention is to provide a microencapsulation preparation method of 5-HT nerve cells induced by MSCs and ASCs. the

The purpose of the present invention is to provide an application of microencapsulated inducible 5-HT nerve cells to overcome the above-mentioned shortcomings and deficiencies in the prior art. the

The invention belongs to the field of biomedicine, namely microencapsulated 5-HT nerve cells induced by self-MSCs and ASCs and a preparation method thereof. the

Therefore, the inventor designed to use culture medium to isolate and culture MSCs or ASCs, then induce them to differentiate into 5-HT neurons, and then microencapsulate them to isolate and eliminate antigens, protect/nutrition/support their long-term survival and secretion role. The differentiated 5-HT nerve cells induced by the method can be adapted to the treatment of depression, especially refractory or severe depression, stress injury diseases and related cancers through microencapsulation. This kind of 5-HT nerve cells induced by microencapsulated self-stem cells can be transplanted according to the number of cell microcapsules given to the corresponding individual according to the number of cell microcapsules released by the cells. Other antidepressants have clear advantages. The method designed and established by the inventor can provide a reliable new technology and method for the preparation of 5-HT cell maturation for clinical use. the

The invention includes: a method for inducing 5-HT nerve cells by bone marrow mesenchymal stem cells (MSCs) or adipose stromal stem cells (ASCs) and a microencapsulation technology for inducible 5-HT cells. the

①Induce the isolated MSCs or ASCs into 5-HT neurons through a series of conditions;

②Use sodium alginate + nano-polylysine-sodium alginate + nano (alginate-Nano--Poly lysine--alginate-Nano, ANano-P-ANano) to make it microencapsulated. the

The technical problem to be solved in the present invention can be realized through the following technical solutions:

As the first aspect of the present invention, the 5-HT nerve cells induced by MSCs and ASCs are characterized in that the MSCs or ASSCs are induced to become 5-HT nerve cells through step-by-step culture using culture medium: comprising the following steps:

(1) MSCs or ASSCs culture and passage:

①Primary culture;

② digestion and passage;

(2) Precursor induction adopts precursor induction solution for cultivation and induction. In the precursor induction solution, cells that have been cultured for 2 days in the third generation are added to the precursor induction solution, and the solution is changed every other day, and cultivated for 4 days;

MSCs were induced to become progenitor cells with protruding cells, cell bodies and nuclei tending to be rounded, and the cells were identified as Nestin-positive;

(3) Functional induction uses functional induction medium for culture and induction. The functional induction medium is added with an isolation layer to mature 5-HT neurons, the medium is changed every other day, and cultured for 6 days to continue to induce the precursor cells into processes, cell bodies and nuclei. Round or polygonal 5-HT nerve cells, cell identification MAP2 (microtubule-associated protein 2), NeuN (specific neuronal nuclear protein), 5-HT, TPH positive, culture medium radioimmunoassay to release 5-HT concentration> 15ng/ml;

(4) Stable performance culture Cultured in nerve cell culture medium for 1 day, the cell identification performance can be stable, and the function of releasing 5-HT is strong, that is, it becomes 5-HT nerve cells induced by MSCs and ASCs. the

Wherein, in step (1), 5-10 ml of bone marrow or 3-5 g of adipose tissue are taken from the primary culture, and the bone marrow is directly counted at a cell density of 1.5×10 ⁶ /ml; the adipose tissue is digested in a small amount first, and then For cell counting, inoculate the cells in a 50ml plastic culture bottle at a cell density of 1.5×10 ⁷ /ml, add DMEM culture solution containing 10% serum, and place in a constant temperature incubator at saturated humidity, 37°C, and 5% CO ₂ nourish;

Isolate and purify MSCs or ASSCs by whole bone marrow or whole adipocyte adherent method: culture the primary cells for 48 hours and change the medium, carefully discard the unattached cells;

After that, the medium was changed every other day, and observed under an inverted microscope until 7 days later, the cells covered the bottom of the side wall of the bottle. the

Wherein, in step (1), the digested and passaged MSCs or ASCs are cultured until the bottom of the bottle is covered with contact inhibition, the culture solution is sucked out, and washed with CMF-PBS; 0.25% trypsin is added, digested at 37°C for 1 min, added Culture solution containing 10% serum to stop the digestion, pipetting to make the cells fall off the bottle wall;

Add 10 ml of 10% serum-containing DMEM culture solution to each bottle, pass passage according to 1:3 bottle, continue to cultivate, and change the medium every other day. the

After 5 days, the bottom of the bottle is covered, digested again and passed to the third generation;

The cells cultured by this method are spindle-shaped, rod-shaped, or multi-spindle-shaped, and CD44 and CD133 positive are identified. the

Wherein, in step (2), the main components of the precursor induction solution are calculated in parts by weight: high sugar DMEM 1000ml, sodium pyruvate 80-120mg, sodium selenite 5-30μg, L-glutamine 250-350mg , 2-mercaptoethanol 3-5μl, NaHCO ₃ 3-5g, HEPES 5-7g, co-inducer basic fibroblast growth factor (bFGF) 100-200ng/brain-derived neurotrophic factor (BDNF) 100-200ng/ All-trans retinoic acid (ATRA) 5 ~ 10mg, / tissue fluid and cytokines 1 ~ 3 parts.

Wherein, in step (3), the main components of the function-inducing liquid (according to parts by weight): high-sugar DMEM 1000ml, sodium pyruvate 80-120mg, sodium selenite 5-30μg, L-glutamine 250- 350 mg, 2-mercaptoethanol 3-5 μl, NaHCO ₃ 3-5 g, HEPES 5-7 g, B275-10 ml, tryptophan 1-5 mg, co-inducer basic fibroblast growth factor (bFGF) 100-200 ng/brain Derived neurotrophic factor (BDNF) 100-200ng/ATRA5-10mg/tissue fluid and cytokine 1-3 parts.

Wherein, in step (4), the main components of the nerve cell culture medium (according to parts by weight): Neuronal Medium 1000ml, B2710-20ml, tryptophan 5-10mg, co-inducible factor is brain-derived neurotrophic factor (BDNF) 100-200ng. the

Wherein, in the step (2) and step (3), the interstitial fluid is insulin 4-8 μ, transferrin 1-5-mg, progesterone 10-50 ng, corticosterone 1-5 mg, cortisol 1-5 mg , 1-5 mg of 3-iodonine, 1-5 mg of biotin, 1 g of human serum albumin, 1-3 mg of putrescine, etc. the

As a second aspect of the present invention, the method for preparing microencapsulation of 5-HT nerve cells induced by MSCs and ASCs is characterized in that it comprises the following steps:

Using the ANano-P-ANano (sodium alginate + nanoparticle-polyamic acid-sodium alginate + nanoparticle) method, the 5-HT nerve cells induced by MSCs and ASCs were microencapsulated by an electrostatic microcapsule generator ;

Each microcapsule contains 20 or 30 5-HT nerve cell groups (Figure 4), so that the cells in the microcapsule can fully contact the wall of the microcapsule, easy to attach and exchange nutrients, and then make microencapsulated inducible 5-HT nerve cells. cell. the

As the third aspect of the present invention, the application of microencapsulated inducible 5-HT nerve cells is characterized in that it is used for preparing medicine for treating depression. the

As the fourth aspect of the present invention, the application of microencapsulated inducible 5-HT nerve cells is characterized in that it is used for preparing anticancer drugs. the

Beneficial effects of the present invention:

The microencapsulated inducible 5-HT cells of the present invention, the microencapsulated human 5-HT nerve cell central transplantation therapy, will provide a new way for clinical treatment of depression and anticancer treatment, and provide experimental tools for other related research Cells, etc., have broad application prospects, which can generate huge economic and social benefits. The characteristics and advantages of microencapsulated inducible 5-HT cells prepared by this method are as follows:

(1) Microencapsulation-induced 5-HT cell transplantation belongs to physiological regulation. the

(2) The inducible 5-HT neurons are not affected by exogenous genes. the

(3) The stem cell induction method is efficient and simple. By using this method, MSCs, MSCs, and ASCs can be induced to become 5-HT nerve cells with a positive rate of more than 95%, and the 5-HT release function is strong. This method only uses the cell exchange medium culture method, which is easy to grasp and operate. the

(4) The prepared microcapsules have good physical properties and can play a role of support/nutrition/protection. the

(5) The prepared microcapsules have good biological stability. the

(6) The prepared microcapsules have suitable micropermeability. the

(7) No immunogenicity. the

(8) Microcapsules of 5-HT nerve cells with good histocompatibility. the

(9) No toxic side effects. the

Description of drawings

Figure 1 shows the 3rd generation MSCs cultured for 5 days

Figure 2 is the induction and culture of MSCs precursors for 4 days

Figure 3A is MSCs induced 5-HT culture for 6 days (toluidine blue staining)

Figure 3B is MSCs induced 5-HT culture for 6 days (5-HT immunohistochemical staining)

Figure 4 is ANano-P-Anano microcapsules

Figure 5 is a schematic diagram of the action principle of microencapsulated inducible 5-HT nerve cells

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. the

The experimental methods that do not indicate specific conditions in the following examples are usually carried out according to conventional conditions, or the conditions provided by the manufacturer. the

The invention includes: a method for inducing 5-HT nerve cells by bone marrow mesenchymal stem cells (MSCs) or adipose stromal stem cells (ASCs) and a microencapsulation technology for inducible 5-HT cells. the

①Induce the isolated MSCs or ASCs into 5-HT neurons through a series of conditions;

②Use sodium alginate + nano-polylysine-sodium alginate + nano (alginate-Nano--Poly lysine--alginate-Nano, ANano-P-ANano) to make it microencapsulated. the

Bone marrow, that is, bone marrow mesenchymal stem cells (bone mesenchymal stem cells, MSCs) was sourced from the Biomedical Tissue Engineering Research Center of Shanghai Second Military Medical University, Shanghai, China. Bone marrow mesenchymal stem cells can be autologous or allogeneic. the

Adipose tissue, that is, adipose stromal stem cells (ASCs) was obtained from the Biomedical Tissue Engineering Research Center of Shanghai Second Military Medical University, Shanghai, China, and the adipose stromal cells could be autologous or allogeneic. the

The microencapsulated inducible 5-HT nerve cells are ready to use. the

Example 1

1. MSCs or ASSCs two-step method to induce 5-HT neurons

Using a series of conditioned medium designed and prepared, MSCs or ASSCs were cultured step by step to induce 5-HT neurons. the

(1) MSCs or ASSCs culture and passage:

①Primary culture: According to the patient’s condition and body weight, take 5-10ml of his own bone marrow or 3-5g of adipose tissue, and directly count the cells of the bone marrow at a cell density of 1.5×10 ⁶ /ml; Perform cell counting, inoculate the cells in 5 to 10 100ml plastic culture bottles at a cell density of 1.5×10 ⁷ to 1.5×10 ⁹ /ml, add DMEM culture solution containing 10% serum, and place in saturated humidity, 37 ℃, 5% _CO2 constant temperature incubator.

Isolation and purification of MSCs or ASSCs by whole bone marrow or whole adipocyte adherence method: primary cells were cultured for 48 hours and the medium was changed, and non-adherent cells were carefully discarded. the

After that, the medium was changed every other day, and observed under an inverted microscope until 7 days later, the cells covered the bottom of the side wall of the bottle. the

② Digest and subculture MSCs or ASCs until the bottom of the bottle is covered with contact inhibition, suck out the culture medium, add CMF-PBS to wash; add 0.25% trypsin, digest at 37°C for 1min, add culture medium containing 10% serum to stop For digestion, the cells were detached from the flask wall by pipetting. the

10 ml of DMEM culture solution containing 10% serum was added to each bottle, and the culture was continued in 1:3 bottles, and the medium was changed every other day. the

The bottom of the bottle was covered in 5 days, and digested again to pass to the third generation. the

The cells cultured by this method are spindle-shaped, rod-shaped, or multi-horizontal (Figure 1), and CD44 and CD133 are identified as positive. the

(2) Precursor induction is cultured and induced by precursor induction solution. The main components of the precursor induction solution are: high-sugar DMEM 1000ml, sodium pyruvate 80-120mg, sodium selenite 5-30μg, L-glutamine 250- 350 mg, 2-mercaptoethanol 3-5 μl, NaHCO ₃ 3-5 g, HEPES 5-7 g, co-inducer basic fibroblast growth factor (bFGF) 100-200 ng/brain-derived neurotrophic factor (BDNF) 100-200 ng / all-trans retinoic acid (ATRA) 5-10mg, / interstitial fluid (the interstitial fluid is insulin 4-8μ, transferrin 1-5-mg, progesterone 10-50ng, corticosterone 1-5mg, cortisol 1 ~5mg, 1-5mg of 3-iodonine, 1-5mg of biotin, 1g of human serum albumin, 1-3mg of putrescine, etc.) and 1-3 copies of cytokines, add the cells cultured for 2 days in the 3rd generation to the precursor The induction medium was changed every other day, and cultured for 4 days.

The MSCs were induced to become progenitor cells with protruding cells and rounded cell bodies and nuclei (Figure 2), and the cells were positive for Nestin. the

(3) Functional induction is cultivated and induced by functional induction medium. The main components of the functional induction medium (according to parts by weight): high-sugar DMEM, pyruvate, L-glutamine, 2-mercaptoethanol, NaHCO ₃ , HEPES, B27, tryptophan, co-inducible factor bFGF/BDNF/ATRA/interstitial fluid (the interstitial fluid is insulin 4-8μ, transferrin 1-5-mg, progesterone 10-50ng, corticosterone 1-5mg, cortisol 1 ~5mg, 1-5mg of 3-iodonine, 1-5mg of biotin, 1g of human serum albumin, 1-3mg of putrescine, etc.) and cytokines, etc., add the isolation layer to mature 5-HT nerve cells, change the medium every other day, After culturing for 6 days, the precursor cells were further induced into 5-HT neurons with many protrusions, round or polygonal cell bodies and nuclei. 5-HT, TPH positive, the release of 5-HT concentration>15ng/ml by radioimmunoassay in the culture medium.

(4) The culture with stable performance adopts the culture of nerve cell culture medium. The main components of the nerve cell culture medium are calculated in parts by weight: Neuronal Medium 1000ml, B2710-20ml, tryptophan 5-10mg, co-inducible factor BDNF100-200ng, After culturing for 1 day, the identification performance of the cells was stable, and the function of releasing 5-HT was strong (Fig. 3A and Fig. 3B). the

2. Induced 5-HT neuron microencapsulation

1. Using the ANano-P-ANano (sodium alginate + nanoparticle-polyamic acid-sodium alginate + nanoparticle) method, the 5-HT nerve cells induced by MSCs and ASCs were microencapsulated by an electrostatic microcapsule generator. encapsulation;

Each microcapsule contains 20 or 30 5-HT nerve cell groups (Figure 4), so that the cells in the microcapsule can fully contact the wall of the microcapsule, easy to attach and exchange nutrients, and then make microencapsulated inducible 5-HT nerve cells. cell. the

2. Properties and identification of microcapsule materials:

Experimental observations: ① Microcapsule cells were cultured in a petri dish for 2, 4, and 6 days, and the shape of the peripheral edge of the microcapsule wall was observed under an inverted microscope (no deformation, depression or shrinkage), indicating that the microcapsules had good physical properties and stable biological properties; ② After 2, 4, and 6 days of culture, the 5-HT content in the culture supernatant can reach 8-12 ng/ml, reflecting that the cells survive and function well, indicating that the micro-permeability is suitable; ③ Animal (rat) subarachnoid transplantation There was no rise in body temperature afterwards, indicating that the microcapsules have strong histocompatibility and good isolation, and there is no pyrogen that causes a fever reaction; ④ Histological examination of the spinal cord showed no leukocyte infiltration; centrifugation of the cerebrospinal fluid showed no increase in leukocytes, indicating that these microcapsules have a positive effect on Local non-toxic side effects and so on. the

① Good physical properties; ② Biological stability; ③ Appropriate micro-permeability; ④ Strong tissue compatibility and good isolation; ⑤ No toxic and side effects. the

3. Functional identification of microencapsulated 5-HT nerve cells:

It shows that the microencapsulated nerve cells survive well, and the synthesis and release of 5-HT are vigorous, and the quantum release titer of individual microcapsules and the number of individualized microcapsule cell transplantation of patients are determined. the

For functional testing of the microencapsulated nerve cell group, 1×10 ³ to 5×10 ³ microencapsulated cell groups were inoculated into each 50ml culture bottle, and the supernatant was taken every other day to detect that the 5-HT content was 5~18ng. According to the needs of central regulation, homeostasis balance and compensation function, the individual transplantation volume is 3×10 ⁴ -10×10 ⁴ microcapsules.

3. Principle of action of microencapsulated inducible 5-HT nerve cells

(1) The microcapsules maintain the survival and function of the cells in the capsules, and avoid immune reactions

① The material of cell microcapsules is sodium alginate + nanoparticle-polyamic acid-sodium alginate + nanoparticle 3-layer micromembrane, which can provide suitable attachment and nutrient uptake for cells in the microcapsule, maintain its stability, and improve its The 5-HT synthesis and release function can help the pia mater of the spinal cord in the slow fluid flow area outside the microcapsules to adapt and be compatible with the microcapsules, making it easier to create a survival environment for them;

②The microcapsules provide an isolation barrier for the induced 5-HT cells, preventing them from contacting with immune cells, avoiding the immune response or immune damage caused by them, and maintaining their good survival;

③The microcapsule has suitable permeability, which can make the nutrients outside the capsule easy to penetrate into the capsule to be used by enough cells, maintain the stability of the internal environment in the capsule, and ensure that the released 5-HT is easy to seep out. the

(2) The 5-HT released by the 5-HT cells in the microcapsules compensates for the reduced 5-HT in the cerebrospinal fluid and the depletion of 5-HT in the brain cells, and restores or improves the central nervous system's ability to resist stress damage or tolerance. the

Depression, or stress-related patients or some cancer patients have depletion or low levels of central 5-HT, and central 5-HT depletion may be caused by stress-induced 5-HT cell failure (acquired weakening), Or born with 5-HT cell TPH polymorphism and high susceptibility to stress injury (congenital deficiency), causing its own 5-HT cells to gradually develop into stress tolerance incompetence and decompensation The pathological phenomena of the neurons, manifested as anxiety, irritability, ... etc., which cause the abnormal function of the neurons regulated by the central 5-HT, and lead to the susceptibility of the organ cells controlled by the descending conduction pathway, or the development of pathological malignancy. the

As shown in Figure 5, the microencapsulated inducible 5-HT nerve cells were transplanted into the subarachnoid space 1, and the 5-HT released by the 5-HT cells in the capsule penetrated into the subarachnoid space, and the transplanted 5-HT nerve cells The cells release 5-HT and seep into the microcapsules into the cerebrospinal fluid2, and spread to the corresponding neurons with the circulation of the cerebrospinal fluid to play its role as a transmitter. After the action, 5-HT is inactivated by reuptake by neurons or plays a nutritional role, and the dysfunction of hippocampus, hypothalamus, and dorsal nucleus of vagus nerve can be improved by physiological regulation4. the

The microencapsulated inducible 5-HT cells of the present invention, the microencapsulated human 5-HT nerve cell central transplantation therapy, will provide a new way for clinical treatment of depression and anticancer treatment, and provide experimental tools for other related research Cells, etc., have broad application prospects, which can generate huge economic and social benefits. The characteristics and advantages of microencapsulated inducible 5-HT cells prepared by this method are as follows:

(1) Microencapsulation-inducible 5-HT cell transplantation belongs to physiological regulation. The microencapsulation-inducible cell central transplantation obtained by this preparation method, the 5-HT that is released to compensate for depletion is a normal neurotransmitter. Compared with the existing antidepressant, stress injury or anticancer drugs, the 5-HT cells induced by such microencapsulation have a better physiological regulation effect without causing adverse effects. the

(2) The inducible 5-HT nerve cells are not affected by exogenous genes. This induction method belongs to chemical induction, without the introduction of exogenous carriers (viruses or liposomes, etc.) and genes, so the obtained cells are transplanted into the body, It will not cause abnormal genome changes due to virus infection or allogeneic gene integration to the body, resulting in pathological changes in transplanted cells or other cells in the body. the

(3) The stem cell induction method is efficient and simple. Using this method, MSCs, MSCs, and ASCs can be induced to become 5-HT nerve cells with a positive rate of more than 95%, and have a strong 5-HT release function. This method only adopts the cell-changing medium culture method, and does not need complex techniques such as vector construction and gene transfection or gene introduction, so it is easy to grasp and operate. the

(4) The prepared microcapsules have good physical properties, provide a suitable space for the cells in the capsule to survive, and can play a role of support/nutrition/protection. After being transplanted into the human body, the gap between the cyst cavity and the capsule wall does not shrink or expand. the

(5) The prepared microcapsules have good biological stability. After being transplanted in vivo, they can maintain their stable shape driven by cerebrospinal fluid circulation or attached to pia mater. the

(6) The prepared microcapsule has suitable micro-permeability. The microcapsule wall is rich in suitable porosity and hydrophilicity, which can allow suitable nutrient components of cerebrospinal fluid or nutrient components in pia capillaries after attachment to penetrate into the capsule and 5 -HT The 5-HT released by the nerve cells leaks out of the capsule, that is, it facilitates the exchange of substances inside and outside the microcapsule. At the same time, it has a good isolation effect on the cells in the capsule and the immune system in the body, and avoids any rejection reaction caused by the transplanted cells. the

(7) No immunogenicity The 5-HT nerve cells induced by microencapsulation are derived from autologous cells, so the 5-HT cells in the capsule itself are autologous cells. Theoretically, the induced cells are not immunogenic or Only very weak immunogenicity, that is, it will not cause rejection of the transplantation in the body, that is, after in vivo transplantation, it will not cause subarachnoid space and systemic inflammatory reactions, and no cerebrospinal fluid or blood leukocytes will increase. the

(8) Good histocompatibility The microcapsules of 5-HT nerve cells adopt ANano-P-ANano's sodium alginate, which has good histocompatibility and rich nanoparticle tissue, which further enhances histocompatibility and makes the transplanted cells The microcapsules are compatible with the tissues in contact with each other, which avoids the foreign body reaction attached to the currently used drugs. the

(9) No toxic and side effects The use of microencapsulated 5-HT nerve cells plays a role through the 5-HT released by them. 5-HT is an essential neurotransmitter for the maintenance and regulation of central function, and can also promote the proliferation and growth of neural stem cells Development, increasing or supplementing the required amount of 5-HT for the central nervous system, can play a good physiological role without any toxic side effects, that is, no toxic reactions such as fever, liver, kidney, and nervous system after transplantation. the

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and that described in the above-mentioned embodiments and the description only illustrates the principles of the present invention, and the present invention also has various aspects without departing from the spirit and scope of the present invention. Variations and improvements all fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents. the

Claims (3)

1. a method of utilizing MSCs induction 5-HT neurocyte, is characterized in that, adopting nutrient solution that MSCs is cultivated to induction through substep becomes 5-HT neurocyte, comprises the following steps:

(1) MSCs cultivates and goes down to posterity:

1. former culture;

2. had digestive transfer culture;

(2) precursor induction adopts precursor induced liquid to cultivate and induction, and the 3 cultures cell of 2 days is added to described precursor induced liquid, the next day change liquid, cultivate 4 days;

By the precursor cell that MSCs induces into that projection attenuates, cell space and karyon are tending towards becoming circle, cell identifies that Nestin is positive;

The main component of described precursor induced liquid is according to parts by weight meter: DMEM in high glucose 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO ₃3～5g, HEPES5～7g, the helper-inducer factor are Prostatropin bFGF100～200ng/ Brain Derived Neurotrophic Factor BDNF100～200ng/ all-trans retinoic acid ATRA5～10mg,

(3) function induction adopts function induced liquid to cultivate and induction, add the ripe 5-HT neurocyte of sealing coat, the next day change liquid, cultivate 6 days, by precursor cell continue to induce into that projection is many, cell space and karyon become circle or polygonal 5-HT neurocyte, cell identifies that microtubule-associated protein 2MAP2, specificity neuronal kernel albumen NeuN, 5-HT, TPH are positive, nutrient solution ria-determination its discharge 5-HT concentration >15ng/ml;

The main component of described function induced liquid is according to parts by weight meter: DMEM in high glucose 1000ml, Sodium.alpha.-ketopropionate 80～120mg, Sodium Selenite 5～30 μ g, L-glutaminate 250～350mg, 2 mercapto ethanol 3～5 μ l, NaHCO ₃3～5g, HEPES5～7g, B275～10ml, tryptophane 1～5mg, the helper-inducer factor are Prostatropin bFGF100～200ng/ Brain Derived Neurotrophic Factor BDNF100～200ng/ATRA5～10mg,

(4) stable performance is cultivated and is adopted neuronal cell cultures liquid to cultivate 1 day, and cell is identified stable performance, and the function of release 5-HT is vigorous, becomes the 5-HT neurocyte of MSCs induction,

The main component of described neuronal cell cultures liquid is Brain Derived Neurotrophic Factor BDNF100～200ng according to parts by weight meter: NeuronalMedium1000ml, B2710～20ml, tryptophane 5～10mg, the helper-inducer factor.

2. the method for utilizing MSCs induction 5-HT neurocyte according to claim 1, is characterized in that, in step (1), described former culture is got marrow 5～10ml, and directly cell counting, with 1.5 * 10 ⁶the cell density of/ml; Cell is inoculated in 100ml plastic culture bottle, is contained the DMEM nutrient solution of 10% serum, be placed in saturated humidity, 37 ℃, the interior cultivation of 5%CO2 constant incubator;

With full medullary cell adherent method isolation and purification MSCs: primary cell culture 48h changes liquid, and carefulness discards not attached cell;

After this next day, change liquid, under inverted microscope, observe, at the bottom of covering with bottle sidewall to 7 days cells.

3. the method for utilizing MSCs induction 5-HT neurocyte according to claim 1, is characterized in that, in step (1), described had digestive transfer culture MSCs reaches contact inhibition at the bottom of being cultured to and covering with bottle, and sucking-off nutrient solution, adds CMF-PBS to rinse; Add 0.25% pancreatin, be placed in 37 ℃ of digestion 1min, add the nutrient solution containing 10% serum to digest with termination, suction pipe piping and druming makes cell come off from bottle wall;

Every bottle adds the DMEM nutrient solution 10ml containing 10% serum, by 1:3 bottle sub-bottle, goes down to posterity, continue to cultivate, the next day change liquid;

At the bottom of within 5 days, covering with bottle, 3 generations of had digestive transfer culture to the again;

This method cultured cells is fusiformis, the shaft-like or shape of dashing forward more, identifies that CD44, CD133 are positive.