Infection And Latency Of Hsv-1 On Rat Bone Marrow Mesenchymal Stem Cells Vitro

Objective:To culture bone marrow mesenchymal stem cells (BMSCs) of rat in vitro, the best condition for isolation and culture of bone marrow mesenchymal stem cells was successfully established and some biological characteristics were observed. It found a base for further differentiation and using of bone marrow mesenchymal stem cells.HSV-1 infected BMSCs, to observe HSV-1 infection and the latent infection on BMSCs. So it could reveal effects from HSV-1 on BMSCs.Method:Under the asepsis condition, bone marrow was extracted from the femoral and shin bone of rat, BMSCs were separated from bone marrow and cultivated in two groups, that is DMEM media group and DMEM with mineralized liquid group respectively. To monitor the activity of alkaline phosphatase (ALP) of passage 3 BMSCs by means of enzyme kinetics and to observe BMSC-derived calcification nodule by staining with alizarin. BMSCs were identified by alizarin red staining and detection of ALP. HSV-1 infected BMSCs and CPE was observed. HSV-1 latent infection in BMSCs was established. The total DNA was isolated from HSV-1 infected BMSCs and the specific gene fragment of HSV-1 was amplified by PCR.Result:ALP level in both groups increased gradually. After independent samples t test showed the ALP activity in the DMEM with mineralized liquid group on the 3rd, 6th,9th day was significantly higher than that in DMEM media group(P<0.05). After BMSCs were induced by mineral-fluid for 14 days, BMSCs have manifestated the characteristics of osteoblast and could form calcification nodule. The induced BMSCs have manifested the characteristics of osteoblasts. HSV-1 infected BMSCs showed the typical cytopathic effect and a 477bp fragment of HSV-1 was amplified by PCR. CPE couldn't be found from BMSCs latently infected by HSV-1 when BMSCs were cultured until passage 7th. But the 477bp fragment was still detectable. Our research showed that HSV-1 can establish latent infection in BMSCs.Conclusion:Rat BMSCs can proliferate well under suitable nutrient condition, and can be induced to differentiate into osteoblasts in vitro as well.Therefore they can be used as the seed cells for the bone tissue engineering. Indeed, BMSCs are easily to obtain and cultivate with hypo-immunogenicity and convenient for transfering and expression of exogenous gene. Therefore, BMSCs have been generally applied in cell therapy and gene therapy for clinical diseases as an ideal vector. HSV-1 can infect rat BMSCs and develop the latent infection in vitro. This study could offer foundation to the situations that BMSCs have been used for cell therapy and gene therapy and Organ transplantation clinically. It will guide clinical therapy and improve post transplant patients'quality of life and survival rate.