Isolation And Cultivation Of Swine Bone Marrow Stromal Cells And Study Of Its Differentiation Into Chondrocytes

Bone marrow mesenchymal stem cells (BMSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate into a variety of cells such as osteocytes, chondrocytes, adipocytes, musle cells, neurons tendon cells and ligament cells. Due to the characteristics of sufficient resource, being conveniently derived and having high reproductive activity,BMSCscan be widely amplified in vitro without losing its multi-directional differentiation potentiality. Moreover, when used in allotransplantation, the cells activated minor immunological rejection. Therefore,BMSCs have become an important kind of widespread used seed cells. In order to obtain sufficient BMSCs from pigs, an effective separation method was established. Through the in vitro culture and biological identification, cells isolated from pigs were verified asBMSCsand had the ability of osteoblastic differentiation. In this study, recombinant plasmid pEGFP-C1-TGF-β1 was transfected intoBMSCsin vitro and cells were cultured in conditioned medium and were induced into chondrocytes, providing a new strategy to the research of bone injury repair and bone tissue engineering. Preliminarily findings were as follows:1. Femoral head was aseptically cut out from pigs, bone marrow cavity was broken down, and bone marrow fluid was extracted. Cells sedimentum was suspended and inoculated into culture dish, and were cultured at 37℃in a 5 % CO2 incubator. The primary cells adhered to the dish bottom on a monolayer, presented fibroblast-like morphous. The immunochemical identification of CD44 factor was positive, cells were induced by conditioned medium to differentiate into osteogenic cells, ALP dyeing result was positive and mineralized nodules were formed. Combined with morphocytology observation, the cells isolated and cultured were determined toBMSCs. At the beginning, cells showed long fusiform, and had a high proliferation rate, and the passage cycle is shortened. After the 7th or 8th passage, cells extended to become magnanimous, flat and thin, and proliferated slow. And the passage cycle was extended, cell particulate matters increased as well.2. RNA was extracted from peripheral blood lymphocyte of pigs, and the TGF-β1 gene was amplified, and then was cloned into eukaryotic expression vector pEGFP-C1 which included a green fluorescent report gene. 3.BMSCswere transfected with pEGFP-C1-TGF-β1 by Liposome-mediated method. The distribution and location of pEGFP-C1-TGF-β1 fusion protein was observed with fluorescence inverted microscope. Results showed that green fluorescence was detected in transfected cells, and TGF-β1 expression was positive by indirect immunofluorescence detection. Transfected cells lost the typical fibroblast-like morphous ofBMSCs, while showed triangle and polygon morphous. Immunohistochemistry detection of typeⅡcollagen showed thatBMSCsexpressed typeⅡcollagen, the specific surface marker of chondrocyte.In conclusion, on the basis of the successful and mature techniques ofBMSCsisolation and cultivation,BMSCswere transfected with TGF-β1 and cells differentiated fromBMSCsexpressed the surface marker of chondrocyte, providing a new strategy to gene therapy of cartilage injury repair.