ObjectiveTo observe transforming growth factor β3 (TGF-β3) gene expression and the chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) after TGF-β3 gene is transfected into BMSCs of Diannan small-ear pig. MethodsRecombinant adenovirus 5 (rAd5) was extracted as gene vector and packed into recombinant adenovirus rAd5-TGF-β3, double enzyme digestion and PCR identification were performed. BMSCs were isolated and cultured from bone marrow of 2-month-old Diannan small-ear pigs (weighing, 12-15 kg), and the 2nd generation of BMSCs were harvested for experiments. The experiments were divided into 3 groups. BMSCs were transfected with rAd5-TGF-β3 as experimental group and with empty vector as control group, and non-transfected BMSCs were used as blank control group. The transfection efficiency of exogenous gene was identified by flow cytometry, TGF-β3 protein expression by immunofluorescence and Western blot. The cell morphology of experimental group was observed by inverted phase contrast microscope, and the expression of collagen type II in each group was detected by Western blot. ResultsThe rAd5-TGF-β3 recombinant adenovirus was successfully constructed and transfected into BMSCs. Green fluorescence was observed by immunofluorescence microscope. Flow cytometry test showed the best transfection at 72 hours (transfection efficiency of 84.86%). Immunofluorescence staining showed that the expression of TGF-β3 protein was obvious at 72 hours; Western blot showed that there was a TGF-β3 positive band with a relative molecular mass of 30×103, while the control group and blank control group had no positive band. Obvious chondrogenic differentiation was observed in the experimental group after transfection in vitro, while the control group and blank control group had no obvious chondrogenic differentiation. Western blot showed that there was collagen type II positive band with a relative molecular mass of 130×103 at 21 days after culture, while the control group and blank control group had no positive band. ConclusionrAd5-TGF-β3 gene can be successfully transfected into BMSCs via adenovirus vectors, and stable expression of TGF-β3 protein can be observed, enhancing BMSCs differentiation into chondrocytes, which may provide an experimental basis for gene therapy of joint cartilage defects.
ObjectiveTo compare the recovery of proprioception of the knee after the anterior cruciate ligament (ACL) reconstruction with remnant preservation or not. MethodsBetween January 2010 and October 2012, 40 patients with ACL rupture were divided into remnant preservation reconstruction group (trial group, n=20) and traditional reconstruction group (control group, n=20). There was no significant difference in gender, age, disease duration, injury causes, preoperative Lysholm scores, and preoperative International Knee Documentation Committee (IKDC) scores between 2 groups (P>0.05). All the patients received ACL single-bundle reconstruction surgery with autologous hamstring tendon transplantation under arthroscope. After operation, the function of knee was assessed by Lysholm and IKDC scores and the proprioception was assessed by joint position sense (JPS) value which was evaluated by passive repeat angle test with isokinetic test system. ResultsAll incisions healed by first intention in 2 groups. The patients were followed up 12-16 months (mean, 14.0 months) in trial group, and 12-15 months (mean, 14.5 months) in control group. At 12 months after operation, the Lysholm and IKDC scores were significantly increased when compared with preoperative scores (P<0.05) in both groups, but no significant difference was found between 2 groups (P>0.05). At 3 months and 12 months after operation in trial group, the JPS values of operated knee at 15, 45, and 75° of flexion were significantly lower than preoperative values (P<0.05), but no significant difference was found between at 3 months and at 12 months after operation (P>0.05). At 3 months after operation in control group, there was no significant difference (P>0.05) in JPS values of operated knee at 15, 45, and 75° of flexion when compared with preoperative ones; but at 12 months after operation in control group, the JPS values of operated knee at 15, 45, and 75° of flexion were significantly lower than those at preoperation and at 3 months after operation (P<0.05). At 3 months after operation, the JPS of operated knee at 15, 45, and 75° of flexion in trial group were significantly lower than those of operated knee in control group (P<0.05), but no significant difference was found between 2 groups at 12 months after operation (P>0.05). At 3 and 12 months after operation in trial group, there was no significant difference (P>0.05) in JPS values at 15, 45, and 75° of flexion between operated and normal knees; at 3 months after operation in control group, the JPS values of operated knee at 15, 45, and 75° of flexion were significantly higher than those of normal knee, but there was no significant difference between operated knee and normal knee at 12 months after operation (P>0.05). ConclusionACL reconstruction with remnant preservation is helpful for recovery of proprioception in knee joint at early stage.
ObjectiveTo construct and identify the recombinant adenovirus vector expressing bone morphogenetic protein 2(BMP-2) and transforming growth factor β3(TGF-β3) genes,to observe the expressions of BMP-2 and TGF-β3 after transfected into bone marrow mesenchymal stem cells (BMSCs) of the Diannan small-ear pigs. MethodsBMP-2 cDNA and TGF-β3 cDNA were amplified by PCR,and were subcloned into the pEC3.1(+) plasmid to obtain pEC-GIE 3.1-BMP-2 and pEC-GIE3.1-TGF-β3 plasmid respectively.They were subcloned into pGSadeno vector by homologous recombination reaction and HEK293 cells were transfected after linearization to obtain Ad-BMP-2 and Ad-TGF-β3.The BMSCs were isolated from the bone marrow of Diannan small-ear pig and cultured.The 3rd passage BMSCs were transfered with Ad-BMP-2(group A),Ad-TGF-β3(group B),Ad-BMP-2+Ad-TGF-β3(group C),and untransfected cells served as a control (group D).The expressions of BMP-2 and TGF-β3 genes and proteins were detected by PCR,immunofluorescence,and Western blot.The chondrogenic differentiation of BMSCs was evaluated by immunohistochemical of collagen type Ⅱ. ResultsThe Ad-BMP-2 and Ad-TGF-β3 were constructed successfully and confirmed by PCR and sequencing.The expression clones of Ad-BMP-2 and Ad-TGF-β3 were packaged into maturated adenovirus successfully,the titer was 5.6×108 and 1.6×108 pfu/mL respectively.The PCR results showed a light band at 310 bp in group A and at 114 bp in group B,and both 310 bp and 114 bp bands in group C,but no band in group D.The image of immunofluorescence showed that there were red fluorescence and green fluorescence expressions in the cytoplasm of BMSCs at 72 hours after transfection in groups A and B,respectively;in group C,both red and green fluorescence expressions were detected,and no red or green fluorescence was detected in group D.The results of Western blot showed that there was a light band at 18×103 in group A and at 50×103 in group B;both 18×103 and 50×103 bands were detected in group C;but no band was detected in group D.The cells were positive for collagen type Ⅱ in groups A,B,and C;group C acquired strong collagen type Ⅱ staining when compared with group A and group B;in group D,the cells were negative for collagen type Ⅱ staining. ConclusionThe recombinant adenovirus vector expressing BMP-2 and TGF-β3 are constructed successfully.The BMP-2 and TGF-β3 genes could be expressed effectively in BMSCs of Diannan small-ear pig after transfection,which could afford modified seeding cells for cartilage tissue engineering.