| Background and PurposesOsteonecrosis of femoral head(ONFH)has high disability rate,and prevention and treatment has become a worldwide problem. The occurrence of Steroid-induced ONFH may caused by steroid effects which change some mi RNA expression in the cells, and influence the regulatory effects of relative target gene, thereby change the normal differentiation of BMSCs. The change of the normal differentiation will cause the expression and production of massive adipocytes, and massive reduction in the production of osteoblasts, thereby influence the normal repair of bone cells. So the most important part of the prevention and treatment for Steroid-induced ONFH in this study is to promote the differentiation of osteoblasts and to reduce the differentiation of lipoblasts. Previous studies prevented osteoncerosis by the change of single gene, while this study use the fact that mi RNA have multiple target sites, screen out the Micro RNA-27 a gene which can reduce the differentiation of lipoblasts and promote the differentiation of osteoblasts. This study use the RNA interference(RNA interference, RNI) technology which is more mature now, electroporate the bi-directional control gene mi R-27 a into the outer MSCs of rats, and tests the influence of this gene to the osteoblasts and lipoblasts by multiple testing means and methods such as cytology, biochemistry and molecular biology. This study offers a new idea in mechanism to prevent the occurrence and development of Steroid-induced ONFH. There are no domestic or international studies yet about using mi RNA bi-directional control genes to prevent Steroid-induced ONFH. Research Subjects and MethodsThe study will first choose the SD rats which are 2 to 3 months old, weight are about 180-200 g, then culture and prepare the rat BMSCs by the bone marrow cell obtained from executed rat in aseptic condition. The experiment will be in four groups, the first group is Micro RNA-27 a group(mi RNA1,M):electroporate the bi-directional control gene Micro RNA-27 a into the BMSCs of rats and add the steroid-induction. The second group is the negative control group(negative control, NC): electroporate the invalid sequence genes into the BMSCs of rats and add the steroid-induction into the culture medium. The third group is steroid model group(Model, M): add the steroid-induction into BMSCs only. The last group is normal control group(Normal, N): culture the BMSCs in normal way without any special handling. The experiment will maintain the DXM concentration as 10-7mol/L for the groups which need the steroid-induction, and there will be routine observations for the morphology and growth situation of the cells.The time of first medium change after passage will be recorded as day 0, and RT-q PCR technology will be used separately to measure the relative expression of PARγ, m RNA, BMP and m RNA in each group at day 3 and day 7 after the steroid-induction. At day 14, the ELISA test kit will be used to measure the secretion levels of osteocalcin in the supernates of each group, and the triglyceride contents in the lysised cells. The immunohistochemistry method will be used in cells of each group, and the PPAR BMP protein expression levels in each group will be record and analyzed. At day 21, oil red O method will be used for cell staining in each group, and inverted microscope will be used to observe and record the number of adipocytes. Results 1.Determination of m RNA expression of PPARγ and Runx2:On the 3rd and 7th day of steroid inducement, the expression level of PPARγ m RNA of group M and NC were higher than that of group N(P<0.05), the expression level of PPARγ m RNA of group mi R-27 a were lower than that of group NC and group M(P<0.05).The expression level of Runx2 m RNA of group M and NC were lower than that of group N(P<0.05), the expression level of Runx2 m RNA of group mi R-27 a were almost the same as group N. The difference had no statistical significance(P>0.05). 2.Determination of the amount of adipocytesOn the 14 th day of steroid inducement, BMSCs were stained with oil red O.the content of TG were determined,Group M and NC had greater amount of TG than group N. The difference had statistical significance(P<0.05). Group mi R-27 a had lesser amount of TG than group M and group NC(P>0.05), almost the same amount of TG as group N. The differences had no statistical significance(P>0.05). 3.Determination of OC in the media and the content of TG in osteogenic differentiation of MSCs:On the 14 th day of steroid inducement, group M and group NC had lower OC in the media than that of group N. And Group M and Con had greater amount of TG than group N.The difference had statistical significance(P<0.05). Group mi R-27 a had a bit higher TG in osteogenic and a bit lower OC in the media than group N. The differenceshad no statistical significance(P>0.05). 4.The PPARγ 、 BMP protein expression levels will be record with the im-munohistochemistry method.On the 14 th day of steroid inducement,the MIOD of PPARγ m RNA of group M and NC were higher than that of group N(P<0.05), the expression level of PPARγ m RNA of group mi R-27 a were lower than that of group NC and group M(P<0.05).The MIOD level of Runx2 m RNA of group M and NC were lower than that of group N(P<0.05), the expression level of Runx2 m RNA of group mi R-27 a were almost the same as group N. The difference had no statistical significance(P>0.05). Conclusions:1.the mi R-27 a were able to inhibit the expression of PPARγ m RNA, andmaintained the character of differentiation of MSCs into osteoblastic.2.The mi RNA have multiple target sites, we can use the electroporate the bi-directional control gene Micro RNA-27 a into the outer MSCs of rats, This study offers a new idea in mechanism to prevent the occurrence and development of Steroid-induced ONFH. |
PDF Full Text Request