Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
Objective To investigate the feasibility of the complex of the fibrin sealant (FS) and the bone marrow mesenchymal stem cells（MSCs） to createanew cartilage in the nude mice by the issue engineering technique. Methods T he MSCs were isolated from healthy humans and were expanded in vitro. And then the MSCs were induced by the defined medium containing the transforming growth factor β1 (TGF-β1), dexamethasone, and ascorbic acid. The biomechanical properties of the chondrocytes were investigated at 7 and 14 days. The MSCs induced for 7days were collected and mixed with FS. Then, the FSMSCs mixture was injectedby a needle into the dorsum of the nude mice in the experimental group. In the tw o control groups, only FS or MSCs were injected respectively. The specimens were harvested at 6 and 12 weeks，and the ability of chondrogenesis in vivo was inve stigated by the gross observation, HE, Alcian Blue staining, and type Ⅱ collagen immunohistochemistry. Results The MSCs changed from a spindlel ike fibroblastic appearance to a polygonal shape when transferred to the defined medium, and couldbe induced to express the chondrocyte matrix. After an injection of the mixture , the cartilage-like tissue mass was formed, and the specimens were harvested from the mass at 6 and 12 weeks in the experimental group. The tissue mass at 6 we eks was smaller and relatively firm in texture, which had a distinct lacuna structure. And glycosaminoglycan (GAG) and Type II Collagen expressions were detecte d. The tissue mass at 12 weeks was bigger, firmer and glossier with the mature c hondrocytes lying in the lacuna structure. The positive Alcian blue and Collagen II immunohistochemistry stainings were ber at 12 weeks than at 6 weeks. But there was no cartilage-like tissue mass formed in the two control groups. Conclusion This study demonstrates that the fibrin sealant and the bone marrow mesenchymal stem cells can be successfully used in a constructing technique for the tissue engineered injectable cartilage.
Objective To explore the effect of age and gene therapyon the differentiation of marrow mesenchymal stem cells （MSCs） of the rats. Methods MSCs from the young (1-month-old), adult (9-month-old), and the aged(24monthold) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase(ALP), collagen Ⅰ(Col Ⅰ), bone sialoprotein(BSP) and osteopontin(OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. Results There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col-Ⅰ, OPN and BSP amongthe 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice amongthe 3 groups. Conclusion MSCs obtained from the aged ratscan restore their osteogenic activity following human BMP-2 gene transduction, therefore provides an alternative to treating the aged bone disease.
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To construct green fluorescent protein (GFP)/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway.Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that thesuccessfulconstructionoftherecombinantvectorpEGFP-C1/AktandefficienthighexpressionofpEGFP-C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt (plt;0.05). The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group (SCF+pEGFP-C1/Akt) and control group (SCF+pEGFP-C1), plt;0.05. In experiment group， SCF stimulation enhanced expression of Akt and VEGF mRNA and protein (plt;0.01). Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein through PI3/Akt pathway.
Objective To explore the mechanism of mesenchymal stem cells （MSCs） transplantation for chronic hindlimb ischemia in Lewis rats by using cell tracer technique. Methods MSCs were isolated and cultured by using density gradient centrifugation and adherence method respectively， then labeled by 5-bromo-2-deoxyuridine （BrdU）. Eight chronic hindlimb ischemia models of Lewis rats were prepared by using suture-occluded method and then divided randomly to MSCs transplantation group and control group， each group enrolled 4 rats， accepting MSCs transplantation and saline respectively. Then on 7 days and 14 days after transplantation， clinical observation， determination of blood flow， and angiography were performed on rats of the 2 groups. At the same time points after previous tests， rats of the 2 groups were sacrificed to get quadriceps tissues and gastrocnemius tissues to perform HE staining and BrdU immunohis-tochemistry. Results The 8 rats were all survived on 14 days after transplantation， with no tumor happened and necroses in the transplanted area. On 14 days after transplantation， the blood flow ratio of operated side to un-operated side in the hindlimb （1.773 vs. 1.279） of rats in MSCs transplantation group and control group increased， and the angiography results showed that there were no much increase in ratio of collateral vessels number （0.908 vs. 0.835）. There were no significant change in the quadriceps tissues and gastrocnemius tissues by HE staining. The BrdU positive kernels located in the inter-stitial substance cells and vascular endothelia cells， and divided differently in different parts of hindlimb at different time points， that the ratio of positive cells in gastrocnemius tissue was higher than those of quadriceps tissue on 7 days after transplantation， but lower on 14 days. Conclusions MSCs transplantation can increases the blood perfusion of hindlimb in the early stage of chronic hindlimb ischemia model， and the possible mechanism may be the paracrine effect of MSCs but not the number increase of collateral vessels.
Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
Abstract： Objective To construct a nesprin-siRNA lentiviral vector（LV-siNesprin）, transfect it into bone marrow mesenchymal stem cells （MSCs）, and observe morphology changes of MSCs. Methods According to the target gene sequence of nesprin, we designed and synthesized four pairs of miRNA oligo, which were then annealed into double-strand DNA and identified by sequencing. MiRNA interference with the four kinds of plasmids （SR-1,SR-2,SR-3, andSR-4） were transfected into rat vascular smooth muscle cells, and reverse transcriptase chain reaction(RT-PCR) and Western blotting were performed to detect the interference effects and filter out the most effective interference sequence. We used the best interference sequence carriers and pDONR221 to react together to get the entry vectors with interference sequence. Then the objective carrier pLenti6/V5-DEST expressing both entry vectors and lentiviral vectors was restructured to get lentiviral expression vector containing interference sequence （LV-siNesprin＋green fluoresent protein （GFP））, which was packaged and the virus titer was determined. LV-siNesprin＋GFP was transfected to MSCs, and the expression of nesprin protein（LV-siNesprin＋GFP group，GFP control group and normal cell group）was detected by Western blotting. The morphology of MSCs nuclear was observed by 4’,6-diamidino-2-phenylindole （DAPI） stain. The proliferation of MSCs （LV-siNesprin＋GFP group，GFP control group and normal group） was detected by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide（MTT） after lentivirus transfected to MSCs at 24, 48, 72, and 96 hours. Results The four pairs of miRNA oligo were confirmed by sequencing. Successful construction of LV-siNesprin was confirmed by sequencing. The best interference with miRNA plasmid selected by RT-PCR and Western blotting was SR-3. Lentiviral was packaged, and the activity of the virus titer of the concentrated suspension was 1×106 ifu/ml. After MSCs were transfected with LV-siNesprin, nesprin protein expression significantly decreased, and the nuclear morphology also changed including fusion and fragmentation. The proliferation rate of MSCs in the LV-siNesprin＋GFP group was significantly slower than that of the GFP control and normal cell groups by MTT. Conclusion Nesprin protein plays an important role in stabilizing MSCs nuclear membrane, maintaining spatial structure of MSCs nuclear membrane，and facilitating MSCs proliferation.