Study On Using Gene Knockout Pig Cartilage As Human Cartilage Repairing Material

To solve the difficulty of organ transplantation donor sources,there are many reports on the use of knockout of heterologous antigen genes in pigs(which can reduce the immune response of transplantation)as human tissue organ transplant donors.Considering a large number of patients with articular cartilage damage,it is difficult to solve the problem of the source of cartilage or chondrocytes for transplantation.However,the pigs(referred to as three-knockout pigs)that knocked out the genes of GGTA1/B4GalNT2/CMAH(three kinds of heterologous antigens),which can be used as a hypothesis for the repair of human cartilage donors.Research purposes:To evaluate the feasibility of cartilage of GGTA1/B4GalNT2/CMAH knockout pigs for human articular cartilage repair.Three parts are being included:1)Comparing the biological characteristics and the immunogenicity associated with the transplantation between the knockout pig and the wild pig chondrocytes;observing whether the chondrocytes of the knockout pig reduce the immunogenicity while retaining the biological characteristics of chondrocytes;2)Gal antigen deletion model mice can simulate the human immune environment,which can be used to simulate the immune response of the knockout pig cartilage tissue and the wild type pig cartilage tissue after being implanted into the human skin respectively.To evaluate of the immunogenic performance of gene knockout pig cartilage tissue in humans.3)To evaluate whether knocking genes affect the mechanical properties of pig cartilage,and whether the gene knockout pig cartilage tissue mechanics meets the requirements of human cartilage mechanics.Experimental Method:1.Acquiring the GGTA1/B4GalNT2/CMAH gene knockout pig cartilage tissue and wild-type pig cartilage tissue.and isolated and knocking out knockout porcine chondrocytes and wild-type porcine chondrocytes in vitro to observe cell growth and morphology;Cell cytometry was used to identify the antigen expression of knockout porcine chondrocytes and wild-type porcine chondrocytes.2.The Gal antigen-deficient mice were used as model animals,and the three gene knockout pig cartilage tissues and the wild type pig cartilage tissues were implanted into the subcutaneous tissues of the mice respectively as the experimental group(KO group)and the positive control group(WT group).The sham operation group(con group)was used as a negative control;after 4 weeks,the mouse serum was taken to detect and compare the total IgM,IgG antibody content,anti-Gal IgM,IgG antibody level,non-Gal porcine cartilage IgM,IgG antibody level.Serum cytokine levels,spleen lymphocyte typing.3.Using nanoindentation technology to detect the elastic modulus and hardness of human cartilage tissue,gene knockout pig cartilage tissue,and wild type pig cartilage tissue.Experimental results:1.Gene knockout Porcine chondrocytes grow well and have the same cytological behavior and cell morphology as wild-type porcine chondrocytes.Flow cytometry results showed that the positive rate of Gal antigen expression in wild-type porcine chondrocytes was 96.88%,the positive rate of Sd antigen expression was 13.92%,and the positive rate of Neu5Gc antigen expression was 90.93%.The positive rate of Gal antigen expression in gene knockout porcine chondrocytes was 11.89%,the positive rate of Sd antigen expression was 3.82%,and the positive rate of Neu5Gc antigen expression was 46.59%.2.The total IgM and IgG antibody levels in the serum of mice showed no statistically significant difference between the total IgM and IgG antibody levels in the WT material-implanted group and the total IgM and IgG antibody levels in the KO group.(P>0.05,but there is an increasing trend).The expression levels of anti-Gal IgM and IgG antibodies in the three-knockout pig cartilage tissue were significantly different from those in the WT group,and there was no significant difference compared with the negative control group.On the indicator of serum-specific IgM antibody,there was no significant difference between the con group and the KO group.In the serum-specific IgG antibody index,the con group was significantly different from the KO group.Serum cytokine test results showed that IL-4,IFN-y,IL-10 and IL-12p70 were not significantly different between the con group,the WT group and the KO group(P>0.05).The spleen lymphocyte typing results showed that there was no significant difference between CD group and CD8 T cells between KO group and WT group(P>0.05,but there was an increasing trend).3.There was no significant difference in human cartilage,KO pig cartilage,and WT pig cartilage in terms of elastic modulus and hardness.Experimental conclusions:1.The biological characteristics of gene knockout porcine chondrocytes were not affected by gene knockout;gene knockout porcine chondrocytes showed lower levels of Gal antigen,b4GalNT2 antigen and Sd antigen than wild type porcine chondrocytes.2.Heterogeneous cartilage tissue in the subcutaneous will cause an immune response,gene knockout pig cartilage tissue under the same conditions caused by the immune response is smaller than the wild-type pig cartilage tissue.3.the mechanical properties of gene knockout pig cartilage tissue and human cartilage,wild type pig cartilage at the same level,did not affect the mechanical properties due to gene knockout,can meet the mechanical requirements of tissue transplantation.4.Gene knockout of pig cartilage tissue has the potential for human cartilage repair and deserves further study.