CN111420034A - Mixed stem cell preparation for treating psoriasis and preparation method thereof - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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Abstract
The invention discloses a mixed stem cell preparation for treating psoriasis and a preparation method thereof. The mixed stem cell preparation acts on the psoriasis affected part in a smearing or superficial epidermal injection mode, treats the psoriasis by promoting the regeneration of skin tissues and regulating inflammatory environment, exerts different disease treatment mechanisms on mesenchymal stem cells and hematopoietic stem cells, is safe, efficient and small in side effect, is simple in material taking and production process, and has the potential of realizing medical transformation.
Description
Technical Field
The invention relates to a stem cell medicament with an immunoregulation function, in particular to a mixed stem cell preparation for treating psoriasis and a preparation method thereof.
Background
Psoriasis is commonly called as psoriasis, is a chronic inflammatory skin disease, is a chronic inflammatory reaction skin disease which is mainly characterized by abnormal activation and infiltration of Th1 and Th17 lymphocytes, the pathological changes of the chronic inflammatory reaction skin disease are mainly symptoms of thickening, scaling and erythema of the skin, hyperproliferation of keratinocytes, generation of new blood vessels, infiltration of inflammatory cells and increase of inflammatory factors of an I L23/I L17 inflammatory axis.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a mixed stem cell preparation for treating psoriasis, which can solve the problem that the medicines in the prior art cannot fundamentally relieve the psoriasis symptoms.
In order to solve the problems, the invention adopts the following technical scheme:
in a first aspect of the invention, a mixed stem cell preparation for treating psoriasis is provided, which comprises mesenchymal stem cells, hematopoietic stem cells, human serum albumin and physiological saline.
Further, the mesenchymal stem cells are adipose mesenchymal stem cells, and the hematopoietic stem cells are placental hematopoietic stem cells.
Still further, the adipose-derived mesenchymal stem cells have a density of 4 to 8 by 106Per ml, the density of said placental hematopoietic stem cells is from 0.5 to 8 by 106The mass fraction of human serum albumin is 10-20% per ml.
The Mesenchymal Stem Cells (MSC) are pluripotent cells having the potential of self-replication and multipotentiality, and are originally obtained from bone marrow, have the characteristics of suppressing immune response and inducing peripheral immune tolerance, and have been used for suppressing graft-versus-host reaction caused by bone marrow transplantation, and then, are found in various tissues such as fat, umbilical cord, dental pulp and placenta, and have immune-privileged characteristics, do not express major histocompatibility complex class II molecules, do not express MH I molecules or express MH I molecules at a very low level, and co-stimulatory molecules such as B7-1(CD80), B7-2(CD86) and CD40, CD 40L, and the like, and the deletion of these co-stimulatory molecules results in the loss of secondary signals of effector T lymphocyte activation, T lymphocytes cannot be activated, so that MSC can avoid the attack of T lymphocytes of allogeneic responses by secreting T lymphocytes, whereby MSC transplantation can avoid immune rejection by intercellular direct contact and secretion of cytokines such as PGE 23, O, TGF-734, TNF-T lymphocytes, and cytokines such as T-lymphocyte proliferation suppressive factors, macrophage proliferation and macrophage proliferation inhibitory factors such as monocyte proliferation, macrophage proliferation-activating activity, macrophage proliferation-activating factor, and macrophage proliferation-inhibiting activity, and macrophage proliferation-activating ability of immune receptor proliferation.
The preparation method is used for curing psoriasis, not only is skin damage repaired, but also inflammatory factor infiltration and immune environment abnormality of the affected part of the psoriasis are relieved, and the adipose tissue-derived mesenchymal stem cells have the characteristics of wide source, low immunogenicity, strong amplification capacity and the like, and can regulate and balance an immune system, promote hematopoietic function and repair human tissues and organs.
The placenta contains a large amount of hematopoietic stem cells and immune precursor cells, and the transplantation of the placental hematopoietic stem cells can promote hematopoiesis and remodel the immune system; the placenta hematopoietic stem cell can be clinically transplanted to treat malignant tumor, hemoglobins and abnormal blood diseases, congenital metabolic diseases, congenital immunodeficiency diseases, autoimmune diseases and the like.
In a second aspect of the present invention, there is provided a method for preparing a mixed stem cell preparation, which is used for preparing the above mixed stem cell preparation, and comprises the following steps:
step one, preparing mesenchymal stem cells;
step two, preparing hematopoietic stem cells;
and step three, mixing the mesenchymal stem cells, the hematopoietic stem cells and the human serum albumin in physiological saline according to a proportion.
Further, the mesenchymal stem cell in the first step is an adipose mesenchymal stem cell, and the preparation method comprises the following steps:
A. digesting adipose tissues by using a digestive enzyme solution, and separating a digested cell product;
B. further purifying the cells with a lymphocyte isolate;
C. culturing the purified cells by using a serum-free stem cell culture medium, and culturing the cells in a carbon dioxide cell culture box at 37 ℃ and containing 5% of volume fraction;
D. when the fusion degree of the stem cells is 70 to 80 percent, carrying out cell passage according to the ratio of 1: 3;
E. and collecting the adipose-derived mesenchymal stem cells from the third generation to the sixth generation.
Further, the hematopoietic stem cells in the second step are placental hematopoietic stem cells, and the preparation method thereof comprises the following steps:
a. cleaning placenta with normal saline, cutting placenta lobules with scissors, washing placenta lobules with normal saline, and collecting washing solution;
b. the wash was filtered through a cell strainer and the cells were collected, resuspended in saline, and mixed with 6% hydroxyethyl starch at a ratio of 4: 1, standing for 1 hour at 4 ℃, and removing supernatant to obtain the placenta hematopoietic stem cells.
The mixed stem cell preparation acts on the psoriasis affected part in a smearing or superficial epidermal injection mode, treats the psoriasis by promoting the regeneration of skin tissues and regulating inflammatory environment, exerts different disease treatment mechanisms on mesenchymal stem cells and hematopoietic stem cells, is safe, efficient and small in side effect, is simple in material taking and production process, and has the potential of realizing medical transformation.
Drawings
FIG. 1 is a diagram showing the respective regulation and control abilities of adipose-derived stem cells and mixed stem cells on lymphocytes in an in vitro lymphocyte co-culture experiment.
Detailed Description
Example 1
A mixed stem cell preparation for treating psoriasis comprises mesenchymal stem cells, hematopoietic stem cells, human albumin and normal saline. The mesenchymal stem cells are adipose mesenchymal stem cells with a density of 4 by 106Per milliliter; the hematopoietic stem cells are placental hematopoietic stem cells with a density of 2 by 106Per milliliter; the mass fraction of human serum albumin is 15%.
The preparation method comprises the following steps:
step one, preparing adipose-derived mesenchymal stem cells
A. Transferring the adipose tissues into a 250 ml centrifuge tube, and then adding an equal volume of physiological saline; shaking the centrifuge tube for several times, standing until the adipose tissue floats on the physiological saline, sucking away the physiological saline, adding fresh physiological saline again, and washing fat for several times by the same operation until the blood in the fat is washed away.
B. Digesting adipose tissues by using a DMEM medium containing 0.15% of collagenase I, 0.15% of collagenase II and 1/2 × Accutase/digestive enzyme, digesting the adipose tissues in a constant temperature shaking table at 37 ℃ and 100 rpm for 15 minutes, adding an equal volume of the 10% FBS-containing medium to neutralize the digestive enzyme after digestion is finished, centrifuging at 400g for 10 minutes, and collecting cell precipitates.
C. The cell pellet was resuspended in saline, and the cells were passed through filters 100 microns and 40 microns in diameter, one after the other, to collect individual cells. Adding 20 ml of lymphocyte separating medium Ficoll into a clean 50 ml centrifuge tube, slowly adding the cell suspension onto the lymphocyte separating medium, closing a speed reducing valve of a centrifuge, centrifuging at the rotating speed of 400g for 30 minutes, collecting a white cell layer between an upper liquid layer and the separating medium, re-suspending the cells by using 2 times of volume of physiological saline, centrifuging at the rotating speed of 300g for 10 minutes, and discarding the supernatant.
D. Washing the cells with physiological saline again, centrifuging to remove supernatant, suspending the cells with fresh medium, and culturing the cells in culture flasks, wherein the adipose derived mesenchymal stem cell culture medium is selected from MesenCult of STEMCE LL Technologies, CanadaTMACF Plus medium, the flask being previously coated with an adherence-promoting factor. After 24 hours of culture, the medium was changed and cells that did not adhere to the cell were washed away with physiological saline.
E. When the fusion degree of the cells reaches 70 to 80 percent, digesting the cells with Tryp L ETM for passage, wherein the passage ratio of the cells is 1:3, and collecting the adipose-derived mesenchymal stem cells from the third generation to the sixth generation.
Step two, preparing placenta hematopoietic stem cells
a. Soaking placenta in normal saline, cutting placenta lobules with sterile scissors, washing placenta lobules with normal saline, and collecting the washing solution.
b. The washing solution was filtered through cell filters with diameters of 100 and 40 microns, respectively, and then transferred to a cell centrifuge tube, centrifuged at 400g for 10 minutes, the supernatant was decanted, the cells were resuspended in physiological saline, and then the cells were mixed with 6% hydroxyethyl starch according to a ratio of 4: 1, standing for 1 hour at 4 ℃, and taking the supernatant; centrifuging for 10 minutes at a rotating speed of 400 g; the supernatant was decanted off and washed with physiological saline again to obtain placental hematopoietic stem cells.
Step three, collecting the cultured adipose tissue-derived mesenchymal stem cells and the purified placental hematopoietic stem cells, and mixing the adipose tissue-derived mesenchymal stem cells and the purified placental hematopoietic stem cells in a certain proportion, wherein the density of the adipose tissue-derived mesenchymal stem cells in the preparation is 4 × 106Per ml, the density of placental hematopoietic stem cells was 2 × 106The concentration of human serum albumin was 15% per ml.
Example 2
A mixed stem cell preparation for treating psoriasis comprises mesenchymal stem cells, hematopoietic stem cells, human albumin and normal saline. The mesenchymal stem cells are adipose mesenchymal stem cells with a density of 4 to 8 by 106Per milliliter; the hematopoietic stem cells are placental hematopoietic stem cells with a density of 0.5 to 8 by 106Per milliliter; the mass fraction of human serum albumin is 15%.
The procedure for preparing the mixed stem cell preparation of this example was the same as in example 1.
Example 3
A mixed stem cell preparation for treating psoriasis comprises mesenchymal stem cells, hematopoietic stem cells, human albumin and normal saline. The mesenchymal stem cells are adipose mesenchymal stem cells with a density of 8 by 106Per milliliter; the hematopoietic stem cells are placental hematopoietic stem cells with a density of 8 by 106Per milliliter; the mass fraction of human serum albumin is 20%.
The procedure for preparing the mixed stem cell preparation of this example was the same as in example 1.
Example 4
A mixed stem cell preparation for treating psoriasis comprises mesenchymal stem cells, hematopoietic stem cells, human albumin and normal saline. The mesenchymal stem cells are adipose mesenchymal stem cells with a density of 4 by 106Per milliliter; the hematopoietic stem cells are placental hematopoietic stem cells with a density of 5 by 106Per milliliter; the mass fraction of human serum albumin is 10%.
The procedure for preparing the mixed stem cell preparation of this example was the same as in example 1.
Example 5
A mixed stem cell preparation for treating psoriasis comprises mesenchymal stem cells, hematopoietic stem cells, human albumin and normal saline. The mesenchymal stem cells are adipose mesenchymal stem cells with a density of 6 by 106Per milliliter; the hematopoietic stem cells are placental hematopoietic stem cells with a density of 4 by 106Per milliliter; the mass fraction of human serum albumin is 15%.
The procedure for preparing the mixed stem cell preparation of this example was the same as in example 1.
In vitro validation experiment
To examine the immunomodulatory function of the mixed stem cell preparation of this patent, we compared the ability of simple adipose derived mesenchymal stem cells and the mixed stem cell preparation of example 1 to modulate immune cells by in vitro lymphocyte co-culture experiments. As can be seen from fig. 1, compared with the simple adipose-derived mesenchymal stem cells, the mixed stem cell preparation has stronger abilities of inhibiting Th1 cells and Th17 cells and promoting Treg cells.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (6)
1. A mixed stem cell preparation for treating psoriasis is characterized by comprising mesenchymal stem cells, hematopoietic stem cells, human serum albumin and normal saline.
2. The mixed stem cell preparation for treating psoriasis as claimed in claim 1 wherein the mesenchymal stem cells are adipose mesenchymal stem cells and the hematopoietic stem cells are placental hematopoietic stem cells.
3. The mixed stem cell preparation for treating psoriasis as claimed in claim 2 wherein the density of adipose derived mesenchymal stem cells is 4 to 8 by 106Per ml, the density of said placental hematopoietic stem cells is from 0.5 to 8 by 106The mass fraction of human serum albumin is 10-20% per ml.
4. A method of preparing a mixed stem cell preparation for use in preparing a mixed stem cell preparation according to any one of claims 1 to 3, comprising the steps of:
step one, preparing mesenchymal stem cells;
step two, preparing hematopoietic stem cells;
and step three, mixing the mesenchymal stem cells, the hematopoietic stem cells and the human serum albumin in physiological saline according to a proportion.
5. The method of claim 4, wherein the mesenchymal stem cell of the first step is adipose mesenchymal stem cell, and the method comprises:
A. digesting adipose tissues by using a digestive enzyme solution, and separating a digested cell product;
B. further purifying the cells with a lymphocyte isolate;
C. culturing the purified cells by using a serum-free stem cell culture medium, and culturing the cells in a carbon dioxide cell culture box at 37 ℃ and containing 5% of volume fraction;
D. when the fusion degree of the stem cells is 70 to 80 percent, carrying out cell passage according to the ratio of 1: 3;
E. and collecting the adipose-derived mesenchymal stem cells from the third generation to the sixth generation.
6. The method of claim 4, wherein the hematopoietic stem cells of step two are placental hematopoietic stem cells, and the method comprises:
a. cleaning placenta with normal saline, cutting placenta lobules with scissors, washing placenta lobules with normal saline, and collecting washing solution;
b. the wash was filtered through a cell strainer and the cells were collected, resuspended in saline, and mixed with 6% hydroxyethyl starch at a ratio of 4: 1, standing for 1 hour at 4 ℃, and removing supernatant to obtain the placenta hematopoietic stem cells.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111840333A (en) * | 2020-07-31 | 2020-10-30 | 曲俊峰 | External anti-inflammation bactericidal composition and preparation method thereof |
| CN116725948A (en) * | 2023-07-14 | 2023-09-12 | 黑龙江八一农垦大学 | Adipose stem cell-mediated gel and its preparation method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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