Experimental Study On Isolation And Culture Of Rat Fibrocyte-derived Stem Cells And Induction Of Chondrogenic Cell Differentiation By Chinese Medicine

Objective:To isolate and identify rat fibrogenic stem cells by the combination of wall attachment method and fibronectin differential adhesion method,to observe the purification effect and differentiation potential,and to explore the biological characteristics of fibrogenic stem cells;to apply Chinese medicine to intervene in the chondrogenic differentiation of fibrogenic stem cells,to screen the Chinese medicine that can effectively promote the differentiation of fibrogenic stem cells to chondrogenic cells,and to provide an experimental basis for the establishment of an effective fibrogenic stem cell culture system and targeted induction system.To provide an experimental basis for the establishment of an effective culture system and targeted induction system for fibrogenic stem cells.Methods:Ten 3-month-old SD rats were used to obtain fibro-ring tissue from intervertebral discs,primary fibro-ring-derived stem cells were obtained by mechanical-enzyme digestion,and fibro-ring-derived stem cells were purified by the apposition method combined with differential adhesion method of fibronectin,and the morphological changes and proliferation of the cells were observed under the microscope,and the surface markers of stem cells were detected by cellular immunofluorescence and Real-time PCR techniques.The 3rd generation fibrocyte-derived stem cells were extracted,and the lyophilized powder solutions of Angelica sinensis,Astragalus membranaceus,Radix rehmanniae,and Cortex tulipifera were prepared with chondrogenic induction culture solution at different concentrations(0.1μg·m L^-1,1μg·m L^-1,10μg·m L^-1,100μg·m L^-1)for chondrogenic induction.chondrogenesis induction.The proliferation of the cells was observed,and the cells were stained and observed using saffron O staining night color,and the gene expression of Collagen-II and Sox-9 was detected by Real-time PCR technique.Results:1.Cell morphology and MTT results:When primary cells were cultured for24h,the cells started to grow against the wall with irregular morphology;by day 4,the cells started to proliferate and the morphology was mainly round and triangular;by day 7,the morphology was mainly polygonal and star-shaped multi-protruding cells;by day 14,the cells were growing well and no obvious changes in morphology were seen,and the cell fusion degree had The MTT results showed that the growth curve of fibrocyte-derived stem cells was roughly S-shaped;2 days before inoculation was the latent growth period,3-8 days into the logarithmic growth period,and 8 days later the cell growth entered the plateau phase.2.Stem cell surface marker detection results:Real-time PCR detection of fibro-ring-derived stem cell surface marker genes showed that,compared with the control group,the fibro-ring-derived stem cell group had higher relative mRNA expression of CD105,CD73and CD90（CD105:P<0.01;CD73 and CD90:P<0.05）,and CD45 and CD34 had lower relative mRNA expression was lower（P<0.05）;cellular immunofluorescence staining showed positive expression of CD105,CD73,CD90 and negative expression of CD45 and CD34,which were specific markers on the surface of fibrocyte-derived stem cells.3.The staining results of the three lines of induced differentiation cells:（1）After osteogenic induced differentiation,the cells were observed to be red-stained under microscopic staining of Alizarin Red S.The stained cells showed a typical osteoblast morphology with obvious red calcium nodule deposits,while the control cells were negative without obvious staining and calcium nodules;（2）After chondrogenic induced differentiation,the cells were observed to be expanded and rounded under microscopic staining of Fancy Red O,with a chondrocyte-like morphology and（3）After chondrogenic induction of differentiation,the cells were observed to be expanded and rounded,with chondrocyte-like morphology and obvious red staining.4.The results of the characteristic genes of the cells after induction of differentiation of the three lines:Compared with the control group:Collagen-I and Runx-2 gene mRNA were highly expressed in the cells after 3 weeks of osteogenic induction culture（P<0.05）;Collagen-II and Sox-9 gene mRNA were highly expressed in the cells after 3 weeks of chondrogenic induction culture（P<0.05）;lipogenic The mRNA of PPAR-γand LPL genes were highly expressed in the cells after 2 weeks of induction culture（P<0.05）.5.MTT assay results of cells after induction of chondrogenic differentiation by traditional Chinese medicine:The results of cell proliferation assay by MTT method showed that at 2 days,only the concentration of 10μg·m L^-1of E.tulipifera had a strong promotion effect on the proliferation of fibrocyte-derived stem cells（P<0.01）;at 4 days,the concentration of10μg·m L^-1of E.tulipifera and Angelica sinensis had a strong promotion effect on the proliferation of fibrocyte-derived stem cells（P<0.01）,and the effect of 10μg·m L^-1concentration of Radix et Rhizoma rehmanniae was slightly weaker（P<0.05）;at 6 days,10μg·m L^-1concentrations of E.tulipata,Radix Angelicae Sinensis,Radix et Rhizoma rehmanniae and 100μg·m L^-1concentration of Radix et Rhizoma rehmanniae all showed strong pro-proliferative effects（P<0.01）);at 8 days,10μg·m L^-1concentrations of E.tulipatae,Radix et Rhizoma rehmanniae,Radix et Rhizoma rehmanniae and Radix et Rhizoma rehmanniae all showed strong pro-proliferative effects（P<0.01）,with the strongest effect of E.tulipifera.6.The results of chondrogenic differentiation induced by the four Chinese herbal medicines were stained with saffron O:After 21 days of induction of chondrogenic differentiation,the cells were expanded and rounded and showed obvious chondrocyte-like morphology,and the red staining of the cells could be seen under the microscope of saffron O staining.7.The results of Real-time PCR assay after induction of differentiation by traditional Chinese medicine:Compared with the control group,the expression of mRNA of Collagen-II gene was increased in 10μg·m L^-1Angelica,Shu Di Huang and Tu Bie Chong groups（Angelica,Shu Di Huang:P<0.01;Tu Bie Chong:P<0.05）;compared with the control group,the mRNA of Sox-9 gene in Angelica,Huang Qi and Tu Bie Chong groups expression was increased（P<0.01）compared with the control group.Among them,Angelica promotes the strongest mRNA expression.Conclusions:（1）The wall-mounted method combined with the differential adhesion method of fibronectin could successfully isolate and purify rat fibrocyclic-derived stem cells,and the isolated cells grew well and had good proliferation ability as well as typical mesenchymal stem cell properties and good multi-directional differentiation potential;（2）Chinese herbal medicines Angelica sinensis,Astragalus membranaceus,Radix et Rhizoma tulipifera,and Rhizoma tulipifera could effectively promote the proliferation of fibrocyclic-derived stem cells,and significantly increase the mRNA expression of chondrogenic characteristic gene markers Collagen-II and Sox-9,which have the ability to promote the chondrogenic differentiation of fibrocyclic-derived stem cells.