| As common diseases in sports and daily life,tendon injury and muscle injury have high incidence and poor healing rates,affecting people’s normal life,especially body function and sports potential of athletes.Stem cells play significant role not only in individual development,but also in repairment of pathological conditions and is increasingly becoming front-burner in regenerative medicine,sports medicine and clinical medicine.The discovery of stem cells and the biological characteristics laid a theoretical foundation for the study of musculotendinous physiological and pathological mechanism,meanwhile,which significantly promoted the research on repair and regeneration of motor system disease.This study mainly established an in vitro amplification and culture system for TDSCs of fetal bovine and pig,skeletal muscle satellite cells of pig,studied the biological characteristics and multi-differentiation potential and simulate model of Collagenase-induced tendon injury model and CTX-induced muscle injury model in mice,respectively,combined which to explore the regenerative potential of cell-based therapy for tendon tissues.The results are showing as follows.1.TDSCs Fetal bovine and pig were isolated and digested by using type Ⅰ collagenase,freshly isolated fetal bovine cells were subcultured up to 43 passages and porcine cells were passaged to 11 generations in vitro.The expression of specific surface markers,such as collagen Ⅰ,collagen Ⅲ,CD40,CD90,CD105 and α-SMA were characterized by immunofluorescence,which were all positively expressed.The expression of mRN A of specific genes of tenocytes,such as collagen Ⅰ,collagen Ⅱ and collagen Ⅲ were detected by RT-PCR(reverse transcription polymerase chain reaction)assays and the results showed that TDSCs were positive for collagen Ⅰ and collagen Ⅲ,but negative for collagen Ⅱ who was spec ific gene of chondrocytes.Their cell growth curve,colony-forming efficiency and Karyotype analysis were also identified.In order to explore the multi-lineage differentiation potential,TDSCs were incubated in inducing medium to differentiate into adipocytes,osteoblasts and chondrocytes and the results were identified by Staining reagent and RT-PCR.These findings showed that tendon-derived stem cells possessed properties of self-renewal,colony-forming capacity and multi-differential potential when cultured in vitro.2.The porcine tibialis anterior muscle was digested by the combination of typeⅠ collagenase and trypsin,orderly.Cells were separated and purified by the method of differential cultivation after seeding for 24 h and finally extended to 12 generations.The specific markers of muscle stem cells,such as Myod,Moygenin,Pax7,c-Met,PCNA and Nanog,were detected by RT-PCR and immunohistochemical assays.In this paper,the biological characteristics of the muscle satellite cells and its multiple differentiation potential were also explored in vitro,respectively.3.CTX-induced muscle injury model was established in mice,the normal tissues of muscle were destroyed and massive necrosis in paraffin pathological biopsy,the amount of muscular related enzymes,such as creatine kinase(CK),lactate dehydrogenase-5(LDH-5)in serum,were shown much more than normal range.After explanation of muscle satellite cells into injured site when marked with CM-Dil,both histology section and serological results showed that the injured muscle recovered to a certain degree.Type I collagenase-induced tendon injury model in mice was established,disorganized collagen and serious tendon matrix damage were obvious in histologic section,TDSCs were marked which cell tracker and then injected into injured tendon tissue,H&E-stained sections and the related serological indicators both showed that the treatment of cell transplantation has achieved a positive repair effect,which indicated TDSCs and muscle satellite cell have therapeutic effect in tendon injury and muscle injury in mice model.In summary,this study introduced the research progress of tendon injury、muscle injury and stem cells,achieved great success in isolation and culture of fetal bovine TDSCs、porcine TDSCs and porcine satellite cells in vitro,and explored their biological c haracteristics and multi-lineage differentiation potential.By cell-based therapy in vitro,stem cells were transplanted to injury site in mice and has stimulated repair at some extent.This paper verified the self-renewal and multipotent capacity of stem cells,which was of great significance to provide preliminarily experimental basis for the clinical application of cell-based strategy to improve tissue repair and regeneration. |
PDF Full Text Request