Establishment Of The Rat Bone Mesenchymal Stem Cell Lines Stably Expressing ChM-I

Objective:To study the further role of ChM-Ⅰin inducing BMSCs into chondrocytes, the expressing plasmid pcDNA3.1(+)/ChM-Ⅰwas constructed, and transfected into the MSCs.We make it expressed stably,and verify that it is upregulated in the MSCs,which could make preparation for studying the further role of ChM-Ⅰin inducing BMSCs into chondrocytes and constructing the tissue engineering .Methods:1.Extract the total RNA of rats cartilage by Trizol . Our group designed the specific primers(forward primer:5'-GCAGACAAGCTTATGACAGAGAACTCGGACA -3',reverse primer:5'-GCAGACGCGGCCGCTTACACCATGCCCAAGATG -3') for the ChM-Ⅰgene and amplified the ChM-Ⅰgene (NO.AF051425)by PCR. The product was acquired by PCR and the pcDNA3.1(+) was digested by the enzymes,and then they were connected directionally with each other by the DNA ligase . The ligation product was used to transform E.coli TOP 10 and the positive clones were identified by being digested, sequencing and comparing with the sequence according to the Gene Bank.2. MSCs were obtained from SD rats using the method of density gradient centrifugation culture. Cell surface antigens were detected by flow cytometry ; To demonstrate the multiple differentiation potential of the MSCs ,MSCs were induced into adipocytes and osteoblasts.3. Transfections were performed using Lipofectamine 2000 .Three groups were divided , which were the experimental group, the control group and the blank group.The experimental group was the one that the MSCs were transfected with the plasmid pcDNA3.1(+)/ChM-Ⅰ,the control one with the plasmid pcDNA3.1(+)and the blank one with none of plasmid .We determined the best selection concentration of G418 to culture the rat MSCs by the pre-test and it is 200ug/ml. Medium would be changed after 6 hours.Then the G418 was used to select the cell strains stably expressing the ChM-Ⅰ.4. Take steady screen cells after three generations of continuous culture and extract samples . The capability of transcription and translation were detected in the level of mRNA and protein respectively in order to identify the stability of the expression of ChM-Ⅰin cell lines by RT-PCR and Western blot.Result:1. The positive clones were identified by PCR and sequencing. The length and the sequencing result were consistent with the information of ChM-Ⅰin the Genebank.2. Medium was changed after 48 hours. Part of the cells were perented for spindle or triangle in the bottom of the dish. The density of cell reached 90% after culturing for 11-13 days. After passage, the density of the cells reached to 80% within 6 hours, and they grew faster than the original generation. Some of the cells were long fusiform,like the fibroblast-like cells, growing densely and trimly and another cells seemed whirlpool-like. The positive rate of the positive surface antigen-CD90 and CD29-of the MSCs was 99.79% and 98.41% respectively, while the negative rate of the negative one-CD45 and CD11b/c-was 2.77% and 2.51% respectively. After two weeks of adipogenic induction, oil red O staining, lipid droplets were orange-red; after three weeks of osteogenic induction, alizarin red staining, red patches were visible under the microscope.3. 6 hours after transfection, it was seen that highly refractive vesicles appeared in a large number of MSCs cell bodies appeared, indicating that the cationic liposomes with plasmid had been transfected into MSCs. After 48 hours, 200ug/ul G418 was added into the medium for selection. 5 days after the selection, a small amout of cells in the blank group floated and died, meanwhile a small amount of cells of the two transfected group died. A larger number of the blank group were dead after 7 days and none of them survived after 14 days of seletion.However, 10% of the two transfected group survived. The survival cells were cultured with the selected medium continually and began to proliferate with accelerating growth rate.After passage ,the cells grew vigorously trimly and whirlpool-like.4. The results of RT-PCR showed : the experimental group, control group and blank group ChM-Ⅰ/GAPDH ratio of the mean gray scale was 1.0817±0.2233,0.3601±0.1260,0.3687±0.0086 . Western blot results: the experimental group, control group and blank group ChM-Ⅰ/GAPDH ratio of the average gray scale was 0.9867±0.0204,0.4155±0.0206,0.3751±0.0081.The results of RT-PCR showed that the level of mRNA of ChM-Ⅰin the experimental group was higher than the control and the blank one . According to Western blot, the quantitative optical density analysis showed that the expression of ChM-Ⅰin the experimental group was significantly higher than the control and the blank one (P <0.05), while it was of no significantly difference(P> 0.05) between the control group and the blank one.Conclusion: The recombinant plasmid pcDNA3.1(+)/ChM-Ⅰwas transfecte into rat bone marrow mesenchymal stem cells. After the selection with G418, MSCs stably expressing ChM-Ⅰwere abtained. This work lays the foundation of studying the further role of the ChM-Ⅰin inducing BMSCs into chondrocytes.