阅读说明：本技术 一种降低gvhd发病率的移植物 (Transplant for reducing GVHD morbidity ) 是由 吴勇君 江良旗 王俊 于 2019-10-21 设计创作，主要内容包括：本发明实施例公开了一种降低GVHD发病率的移植物,其特征在于,所述移植物通过将胎盘亚全能干细胞和脐带血造血干细胞混合培养得到；本发明通过将胎盘亚全能干细胞和脐带血造血干细胞共同培养制备得到移植物,通过将该移植物进行回输,能够有效降低CVHD的发变绿和发病程度。(The embodiment of the invention discloses a graft for reducing the incidence of GVHD, which is characterized in that the graft is obtained by mixed culture of placenta sub-totipotent stem cells and umbilical cord blood hematopoietic stem cells; the invention prepares the graft by co-culturing the placenta sub-totipotent stem cells and the umbilical cord blood hematopoietic stem cells, and the graft is returned, so that the greening and morbidity degree of CVHD can be effectively reduced.)

1. A graft for reducing the incidence of GVHD, wherein the graft is obtained by culturing a mixture of placental sub-totipotent stem cells and umbilical cord blood hematopoietic stem cells.

2. The graft of claim 1, wherein said placental sub-totipotent stem cells and umbilical cord blood hematopoietic stem cells are present in a ratio of (4-6) to 1.

3. The graft according to claim 1, wherein said culture is in particular:

sub-totipotent stem cells of placenta andthe mixed cells of cord blood hematopoietic stem cells are 2 × 10⁶cells/bottle were inoculated, the culture system in each bottle was 15ml of DMDMMEM F12+10ng/ml of EGF +10ng/ml of FGF solution, then the bottle was capped, and the bottle was placed in 5% CO₂And culturing in an incubator with saturated humidity and 37 ℃.

4. The graft of claim 1, wherein said placental pluripotent stem cells are prepared by a method comprising:

(a) selecting and pretreating a placenta amnion;

(b) digesting, filtering, centrifuging and inoculating the washed amnion;

(c) and culturing and amplifying the inoculated cells to obtain the placental sub-totipotent stem cells.

5. The graft of claim 4, wherein said digestion treatment comprises the steps of:

(1) placing the cleaned amnion into a sterile digestion bottle, adding 0.25% trypsin solution with the same volume, mixing uniformly, and standing and digesting in an incubator at 37 ℃ for 5-10 min;

(2) after digestion, adding 0.9% NaCl physiological saline into a digestion bottle to tighten the bottle cap, shaking gently, pouring the mixture in the digestion bottle into a 100mL beaker, clamping amnion by using forceps, washing repeatedly, transferring into a new beaker, adding 50 mL0.9% NaCl physiological saline, washing again, and washing repeatedly for 2-3 times;

(3) transferring the washed amnion into a digestion bottle, adding 0.25% trypsin solution with the same volume, mixing uniformly, and standing and digesting for 15-20min in an incubator at 37 ℃.

6. The graft of claim 4, wherein said inoculation is according to (4-5) x 10⁶The cells/T75 culture bottles are inoculated, wherein the culture system in the T75 culture bottle is 15mLDMEM F12+10ng/ml EGF +10ng/ml bFGF liquid.

7. The graft according to claim 4, wherein said culture expansion comprises the steps of:

(1) inoculating the cells in 5% CO₂Culturing under the conditions of saturated humidity and 37 ℃, changing the liquid in a full amount after culturing for 5-7 days, then changing the liquid in a half amount every 3-5 days, and carrying out passage according to the ratio of 1:3 when 80% of cells are fused;

(2) inoculating the cells into a T75 culture bottle according to a passage ratio of 1:3, wherein the culture system is 15mLDMEM F12+10ng/mL EGF +10ng/mL FGF solution, when the cells grow to 80% fusion degree, removing the upper layer culture solution, sucking 15mL of 0.9% NaCl physiological saline, slightly adding the solution into the culture bottle, and removing the washing solution;

(3) adding 1.5mL of 0.25% trypsin solution into the culture flask, quickly covering the culture flask, lightly beating or shaking left and right, and quickly adding 0.5-1mLFCS to stop the action of trypsin when 80% of cells are single;

(4) adding 10 mL0.9% NaCl physiological saline into the culture bottle after termination, repeatedly blowing and beating by using a PEP gun to ensure that the cells are completely changed into single cells, transferring the single cells into a 50mL centrifuge tube, fully and uniformly mixing, sucking 25 mu L of cell suspension by using a trace sample adding gun, counting the cells, then centrifuging the tube cover, sealing by using a sealing film, and centrifuging at 1000rpm for 5 min;

(5) after the centrifugation, the supernatant was discarded and the number of cells counted was 2X 10⁶Re-inoculating the cell/culture bottle into a new culture bottle, wherein the culture system in the culture bottle is 15mLDMEM F12+10ng/ml EGF +10ng/ml FGF liquid;

(6) place the flask in 5% CO₂Culturing at 37 ℃ under saturated humidity;

(7) passage to P3 generation at 1X 10⁴Inoculating the cells/ml into a 24-well plate, and adding DMEM F12+10ng/ml EGF +10ng/ml FGF solution for culture to obtain P4 generation placental sub-totipotent stem cells.

8. The graft in accordance with claim 1, wherein said cord blood hematopoietic stem cells are prepared by the process of:

1) collecting umbilical cord blood, and adding HESPAN accounting for 20% of the weight of the umbilical cord blood into the umbilical cord blood;

2) mixing the umbilical cord blood added with HESPAN, centrifuging, and removing erythrocytes in the umbilical cord blood;

3) and centrifuging the cord blood without the red blood cells again, and extracting plasma to obtain the cord blood hematopoietic stem cells.

9. The graft of claim 8, wherein in step 2), the centrifugation conditions are 10 ℃ and 50g for 7 min.

10. The graft of claim 8, wherein in step 3), the centrifugation conditions are 10 ℃ and 747g for 15 min.

Technical Field

The embodiment of the invention relates to the technical field of hematopoietic stem cell transplantation, in particular to a graft for reducing the incidence rate of GVHD.

Background

Graft Versus Host Disease (GVHD) is a systemic disease of multiple system lesions (skin, esophagus, gastrointestinal, liver, etc.) that occur after bone marrow transplantation and is one of the leading causes of death. Because of the immunogenetic differences between donors and recipients, immunocompetent cells (mainly T lymphocytes) in transplanted bone marrow recognize different histocompatibility antigens of the recipients and proliferate and differentiate, and after proliferating to a certain extent in the recipients, certain tissues or organs of the recipients are used as target targets to carry out immune attack to generate damage.

A potential lethal clinical complication may occur when alloreactive T lymphocytes are transplanted into immunocompromised patients. Once infused into a recipient, donor T cells recognize host cell antigens, resulting in an immune cascade affecting the liver, gastrointestinal tract, and skin. Currently, methods of treatment and prevention of GVHD include conventional immunosuppression after transplantation, low-intensity pretreatment, and depletion or suppression of T lymphocytes from allogeneic donors prior to transfusion. However, the clinical efficacy of these approaches is limited by a number of side effects. For example, conventional immunosuppression is associated with increased risk of reinfection with viruses and opportunistic viral infections, while low-intensity pretreatment regimens are associated with increased recurrence rates. Currently, depletion or inhibition of donor T lymphocytes is the most promising prophylactic treatment of GVHD. This can be achieved by various methods such as a lymphocytotoxic agent, a specific T-lymphocyte inhibitor, and a T-cell depleting agent selected specifically for C0.9% NaCl saline 34+ hematopoietic stem cells and progenitor cells (HSPCs). Although these methods have been shown to be effective in reducing the incidence of GVHD, they reduce the rate at which the recipient reconstructs the immune system, increase the risk of a patient developing a fatal infection and may have limited graft versus leukemia effects; in addition, the prior art directly transplants the hematopoietic stem cells, and the clinical GVHD incidence is higher.

Disclosure of Invention

Therefore, the embodiment of the invention provides a graft for reducing the incidence rate of GVHD, so as to solve the problem of high incidence rate of GVHD in the prior art.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

according to a first aspect of embodiments of the present invention there is provided a graft for reducing the incidence of GVHD, the graft being obtained by co-culturing placental sub-totipotent stem cells and umbilical cord blood hematopoietic stem cells.

The invention prepares the graft by co-culturing the placenta sub-totipotent stem cells and the umbilical cord blood hematopoietic stem cells, and the graft is returned, so that the greening and morbidity degree of CVHD can be effectively reduced.

Furthermore, the number ratio of the placenta sub-totipotent stem cells to the umbilical cord blood hematopoietic stem cells is (4-6) to 1.

Further, the culturing specifically comprises:

mixing placenta sub totipotent stem cell and umbilical cord blood hematopoietic stem cell according to 2 × 10⁶cells/bottle were inoculated with 15ml of DMDMMEM F12+10ng/ml EGF +10ng/ml bFGF solution in each flask, then the flask was capped, and the flask was placed in 5% CO₂And culturing in an incubator with saturated humidity and 37 ℃.

Further, the placental pluripotent stem cells are prepared by the following method:

(a) selecting and pretreating a placenta amnion;

(b) digesting, filtering, centrifuging and inoculating the washed amnion;

(c) and culturing and amplifying the inoculated cells to obtain the placental sub-totipotent stem cells.

Further, the pretreatment is that the amnion is placed in a small sterile beaker containing 50mL of 0.9% NaCl physiological saline solution, repeatedly washed by a pair of tweezers, taken out and placed in a new sterile beaker, added with 0.9% NaCl physiological saline solution and washed again, so that the amnion is washed by 0.9% NaCl physiological saline solution for more than 3 times, hemolysis is removed, the amnion is taken out by the pair of tweezers and placed on a kidney-shaped plate, the mucous membrane and hemolysis tissues are removed, and the amnion is divided into blocks with the size of 3-4 cm; then the processed amnion is transferred to a new sterile beaker containing 50 mL0.9% NaCl physiological saline solution, and the amnion is repeatedly rinsed, then is put into the new beaker, is added with 0.9% NaCl physiological saline solution for rinsing again, and is repeatedly rinsed for more than 16 times.

Further, the digestion treatment comprises the following steps:

(1) placing the cleaned amnion into a sterile digestion bottle, adding 0.25% trypsin solution with the same volume, mixing uniformly, and standing and digesting in an incubator at 37 ℃ for 5-10 min;

(2) after digestion, adding 0.9% NaCl physiological saline into a digestion bottle to tighten the bottle cap, shaking gently, pouring the mixture in the digestion bottle into a 100mL beaker, clamping amnion by using forceps, washing repeatedly, transferring into a new beaker, adding 50 mL0.9% NaCl physiological saline, washing again, and washing repeatedly for 2-3 times;

(3) transferring the washed amnion into a digestion bottle, adding 0.25% trypsin solution with the same volume, mixing uniformly, and standing and digesting for 15-20min in an incubator at 37 ℃.

Further, after the digestion is finished, 0.9% NaCl physiological saline solution is added and fully shaken, the digested amniotic tissue fluid is poured into a 100mL beaker, the amniotic membrane is taken out by using forceps and is placed into a digestion bottle for repeating the operation for 2-3 times, then the amniotic membrane is discarded by using the forceps, the solution of the obtained digested product is filtered by a 200-mesh filter screen into a 200mL blue-covered bottle added with 20mL of 0.25% trypsin solution, shaken and then is subpackaged into 4 50mL centrifuge tubes.

Further, the centrifugation is to centrifuge the filtrate at 2000rpm for 15 min.

Further, the inoculation is carried out according to a (4-5) multiplied by 106cell/T75 culture flask, wherein the culture system in the T75 culture flask is 15ml DMMEM F12+10ng/ml EGF +10ng/ml bFGF solution.

Further, the culture amplification comprises the following steps:

(1) inoculating the cells in 5% CO₂Culturing under the conditions of saturated humidity and 37 ℃, changing the liquid in a full amount after culturing for 5-7 days, then changing the liquid in a half amount every 3-5 days, digesting by adopting 0.25% trypsin when 80% of cells are fused, and carrying out passage according to the ratio of 1: 3;

(2) inoculating the cells into a T75 culture bottle according to a passage ratio of 1:3, wherein the culture system is 15mLDMEM F12+10ng/ml EGF +10ng/ml bFGF solution, discarding the upper culture solution when the cells grow to 80% fusion degree, sucking 15 mL0.9% NaCl physiological saline, slightly adding the saline into the culture bottle, and discarding the washing solution;

(3) adding 1.5mL of 0.25% trypsin solution into the culture flask, quickly covering the culture flask, lightly beating or shaking left and right, and quickly adding 0.5-1mLFCS to stop the action of trypsin when 80% of cells are single;

(4) adding 10 mL0.9% NaCl physiological saline into the culture bottle after termination, repeatedly blowing and beating by using a PEP gun to ensure that the cells are completely changed into single cells, transferring the single cells into a 50mL centrifuge tube, fully and uniformly mixing, sucking 25 mu L of cell suspension by using a trace sample adding gun, counting the cells, then centrifuging the tube cover, sealing by using a sealing film, and centrifuging at 1000rpm for 5 min;

(5) after the centrifugation is finished, the supernatant is discarded, and the culture medium is re-inoculated into a new culture flask according to the counting result and according to the counting result, wherein the culture system in the culture flask is 15mLDMEM F12+10ng/ml EGF +10ng/ml bFGF liquid;

(6) culturing in 5% CO2 saturated humidity at 37 deg.C;

(7) and (4) passage is carried out until P3 generation, the cells are inoculated into a 24-well plate according to the ratio of 1 × 104 cells/ml, and DMEM F12+10ng/ml EGF +10ng/ml bFGF solution is added for culture, so as to obtain the P4 generation placental sub-totipotent stem cells.

Further, the cord blood hematopoietic stem cells are prepared by the following method:

1) collecting umbilical cord blood, and adding HESPAN accounting for 20% of the weight of the umbilical cord blood into the umbilical cord blood;

2) mixing the umbilical cord blood added with HESPAN, centrifuging, and removing erythrocytes in the umbilical cord blood;

3) and centrifuging the cord blood without the red blood cells again, and extracting plasma to obtain the cord blood hematopoietic stem cells.

Further, in the step 2), the centrifugation conditions were 10 ℃ and 50g, and the centrifugation was carried out for 7 min.

Further, in the step 3), the centrifugation conditions were 10 ℃ and 747g, and the centrifugation was performed for 15 min.

The embodiment of the invention has the following advantages:

the invention prepares the graft by co-culturing the placenta sub-totipotent stem cells and the umbilical cord blood hematopoietic stem cells, and the graft is returned, so that the greening and morbidity degree of CVHD can be effectively reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

Fig. 1 is a microscopic view of the placental pluripotent stem cells prepared in example 1 of the present invention;

FIG. 2 is a graph showing the results of oil red O staining of placental sub-totipotent stem cell mixed cord blood hematopoietic stem cells provided in Experimental example 1 of the present invention;

FIG. 3 is a graph showing the results of oil red O staining of placental pluripotent stem cells provided in Experimental example 1;

FIG. 4 is a graph showing the alizarin red staining result of the placental sub-totipotent stem cell mixed cord blood hematopoietic stem cell provided in Experimental example 2 of the present invention;

fig. 5 is a graph showing alizarin red staining results of placental sub-totipotent stem cells provided in experimental example 2 of the present invention;

FIG. 6 is a report diagram of flow analysis of a mixed culture of placental pluripotent stem cells and umbilical cord blood hematopoietic stem cells prepared according to example 3 of the present invention;

FIG. 7 is a graph showing the annual CVHD mortality rate of a mixed culture of placental sub-totipotent stem cells and umbilical cord blood hematopoietic stem cells infused in Experimental example 4 and a mixed product of stem cells not infused.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.