Objective To observe effects of the direct impaction onthe cell survival and the bone formation of the tissue engineered bone modified by the adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP2) gene and to verify the feasibility of the impacted grafting with it. Methods The marrow stromal cells (MSCs) were separated from the canine bone marrow and were cultured. MSCs were transfected with the Adv-hBMP2 gene and combined with the freeze-dried cancellous bone (FDB) to form the tissue engineered bone. Four days after the combination, the tissue engineered bone was impacted in a simulated impactor in vitro and implanted in the mouse. The cell survivals were evaluated with SEM 1 and 4 days after the combination, immediately after the impaction, and 1 and 4 days after the impaction, respectively. The bone formation and the allograft absorption were histologically evaluated respectively. Results There were multiple layers of the cells and much collagen on FDB before the impaction. Immediately after the impaction, most of the cells on the direct contact area disappearedand there was much debris on the section. Some of the cells died and separatedfrom the surface of FDB at 1 day, the number of the cells decreased but the collagen increased on the surface at 4 days. Histologically, only the fibrous tissue was found in FDB without the cells, the bone formation on FDB was even in distribution and mass in appearance before the impaction, but declined and was mainly on the periphery after the impaction in the AdvhBMP2 modified tissue-engineered bone. Conclusion The simulated impaction can decrease the cells survival and the bone formation of the AdvhBMP-2 modified tissue-engineered bone. The survival cells still function well.It is feasible to use the tissue engineered bone in the impaction graft.
Objective To review the current concepts of gene therapy approachesmediated by adenovirus vectors for bone trauma and bone disease. Methods The recent literature concerned gene therapy mediated by adenovirus vectors was reviewed, which provides new insights into the treatments of bone trauma and bone disease. Results Adenovirus vectors was efficient, achieved high expression after transduction, and could transfer genes to both replicating and nonreplicating cells, such as osteoblasts, osteoclasts, fibroblasts, chondrocytes, bone marrow stromal cells, etc. Gene therapy mediated by adenovirus vectors achieved affirmative results in enhancing bone union and in curing bone diseases, such as osteoporosis and rheumatoid arthritis. Conclusion Gene therapy mediatedby adenovirus offers an exciting avenue for treatment of bone trauma and bone diseases.
Objective To investigate the expression of multigenes mediated by adenovirus in liver cancer cells and the effects on growth of cells transducted with multigenes. MethodsBy construction of recombinant adenovirus containing human p53, B7-1, GM-CSF, and IL-2 genes (Ad-multigenes), the expression level of target genes in three human hepatocellular carcinoma cell lines and a human hepatocellular cell line L02 was detected using ELISA, immunohistochemistry and FACS assay and the change of growth of these cells and the tumor cell apoptosis were observed. Results The human hepatic cells and liver cancer cells were all sensitive to adenovirus infection. At a MOI of 50 PFU/cell, among the cells examined nearly 90% were positive expression and except IL-2, other three genes were expressed with high efficiency. The growth of Ad-multigenes-transduced liver cancer cell lines was inhibited and apoptosis was induced, but the growth of Ad-multigenes-transduced normal hepatic cell line L02 did not change. Conclusion These results indicate that the adenovirus is an efficient vector for gene transfer into human liver cancer cells. These liver cancer cell lines transduced with multigenes constructed on one recombinant adenoviral vector can highly express target genes and their growth was inhibited, and apoptosis appeared.
ObjectiveTo compare the osteogenic effect of bone marrow mesenchymal stem cells (BMSCs) transfected by adenovirus-bone morphogenetic protein 2-internal ribosome entry site-hypoxia inducible factor 1αmu (Ad-BMP-2-IRES-HIF-1αmu) and by Ad-cytomegalovirus (CMV)-BMP-2-IRES-human renilla reniformis green fluorescent protein 1 (hrGFP-1) single gene so as to optimize the source of osteoblasts. MethodsBMSCs were separated and cultured from 1-month-old New Zealand white rabbit. The BMSCs at passage 3 were transfected by virus. The experiment was divided into 4 groups (groups A, B, C, and D) according to different virus: BMSCs were transfected by Ad-BMP-2-IRES-HIF-1αmu in group A, by Ad-CMV-BMP-2-IRES-hrGFP-1 in group B, by Ad-CMV-IRES-hrGFP-1 in group C, and BMSCs were not transfected in group D. The optimum multiplicity of infection (MOI) (50, 100, 150, and 200) was calculated and then the cells were transfected by the optimum MOI, respectively. The expression of BMP-2 gene was detected by immunohistochemistry staining after transfected, the expressions of BMP-2 protein and HIF-1α protein were detected by Western blot method. The osteogenic differentiation potential was detected by alkaline phosphatase (ALP) activity and Alizarin red staining. ResultsThe optimum MOI of groups A, B, and C was 200, 150, and 100, respectively. The expression of BMP-2 was positive in groups A and B, and was negative in groups C and D by immunohistochemistry staining; the number of positive cells in group A was more than that in group B (P ﹤ 0.05). The expression of BMP-2 protein in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). The expression of HIF-1α protein in group A was significantly higher than those in the other 3 groups (P ﹤ 0.05), no significant difference was found among the other 3 groups (P ﹥ 0.05). ALP activity in groups A and B was significantly higher than that in groups C and D (P ﹤ 0.05), group A was higher than group B (P ﹤ 0.05). Calcium nodules could be seen in groups A and B, but not in groups C and D; the number of calcium nodules in group A was higher than that in group B (P ﹤ 0.05). ConclusionThe expression of BMP-2 and osteogenic effect of BMSCs transfected by Ad-BMP-2-IRES-HIF-1αmu (double genes in single carrier) are higher than those of BMSCs transfected by Ad-CMV-BMP-2-IRES-hrGFP-1 (one gene in single carrier).
Objective To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. Methods SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 ± 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. Results SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. Conclusion It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.
Objective To construct adenovirus expressing NGF (Ad-NGF) and to investigate its promotive effect on the reparation and regeneration of sciatic nerve injury in rats. Methods NGF gene sequence was cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK-293 cells, the recombinant adenoviruses-Ad-NGF underwent sequence identification. Thirty-two male SD rats weighing 180-200 g were randomly divided into 4 groups (n=8 rats per group). Sciatic nerve injury model was establ ished by disconnecting and direct suturing the right sciatic nerve in the rat. Theright gastrocnemius muscle of group A and C received Ad-NGF injection and adenovirus vector without NGF gene sequence injection, respectively, and 1 × 108 PFU/per time was given every other day for three times. Group B and D received NGF injection (200 U/d) and normal sal ine (100 ?L/d), respectively, for 3 weeks. The effect of various treatments on injured sciatic nerve was evaluated by performing sciatic nerve function index and nerve electrophysiology detections 31 days after operation. Meanwhile, the sciatic nerve in the anastomosis and at the site 1 cm distal to the anastomosis were obtained, and underwent RTPCR and Western blot analysis for detecting NGF mRNA and protein expression level in the injured sciatic nerve in the rats. Histology, immunohistochemistry, and transmission electron microscope observations were conducted. Results Ad-NGF carrying NGF gene sequence was constructed successfully and confirmed by sequence analysis. The sciatic nerve function index, nerve conduction velocity, evoked potential ampl itude, and latent period of group A was better than those of other groups (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). RT-PCR and Western blot detection: the expression levels of NGF mRNA and protein in group A were greater than those of group B, C, and D (P lt; 0.05), and no significant differences were noted among group B, C, and D (P gt; 0.05). Histology and immunohistochemistry observation showed that the regeneration of the sciatic nerve in group A was obvious superior to that of other groups. Transmission electron microscopy observation suggested there was significant difference between group A and groups B, C, and D in terms of axonal diameter of sciatic nerve cross-section, myel in sheath thickness and nerve fiber number (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). Conclusion Ad-NGF can effectively promote the repair of sciatic nerveinjury in rats, and is a new method for obtaining large amounts of NGF in the area of injured peripheral nerve.
Objective To construct replication-defective adenovirus containing tk gene (ADV-tk). Methods Recombinant adenovirus of ADV-tk was constructed using homologous recombination in cells. After the interested tk gene fragment in the recombinant plasmid obtained was confirmed by PCR, the titre of purified recombinant adenovirus was detected. In vitro study, tk gene in SMMC7721 cells transfected by ADV-tk was investigated by RT-PCR. In vivo study, ADV-tk was injected intraperitoneally into BALB/c nude mice with liver cancer and apoptosis cells in tumor were observed. Results Recombinant adenovirus containing ADV-tk was proved successfully. The titre of purified recombinant adenovirus was 1.4×1010 pfu/ml. In vitro study, tk was integrated and expressed by SMMC-7721 cells. In vivo study, with the injection of ADV-tk, apoptosis cells in tumor increased. Conclusion A replication-defective adenovirus containing tk gene is successfully constructed, which may useful for further research on tumor suicide gene therapy with ADV-tk.
Objective To construct gene silence adenovirus vector targeting both transglutaminase 2 （TG2） and Mer receptor tyrosine kinase （Mertk） synchronously and detect the gene silence function of it. Methods The interfering plasmids targeting TG2 protein and Mertk protein were constructed firstly， then the H1 promoter and RNA interfering （RNAi） sequence were cut and ligated to pAdTrack for constructing pAdTrack/TG2/Mertk. The pAdTrack/TG2/Mertk was transfected into BJ5183 bacterial cells which contained pAdEasy-1， then the plasmid was detected by enzyme digestion after recovery. Adenovirus were harvested after that pAdTrack/TG2/Mertk was infected into HEK293 cells. The virus titer was measured after repeated amplification. The RAW264.7 cells were infected by pAdTrack/TG2/Mertk， pAdTrack/TG2， pAdTrack/Mertk， and pAdTrack/green fluorescent protein （GFP）， respectively. Then the expression levels of TG2 protein and Mertk protein of mouse macrophages were detected by Western blot after infection. Results The virus titer of pAdTrack/TG2/Mertk plasmid was 6.13×1010GFU/mL. The pAdTrack/TG2/Mertk plasmid which contained 2 promoters and 2 RNAi sequences was identified successfully by enzyme digestion. Compared with pAdTrack/GFP group and pAdTrack/Mertk group （there was no significant differece between the 2 groups）， the expression levels of TG2 protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/TG2 decreased obviously （P＜0.01）， but there was no significant difference between the later 2 groups. Compared with pAdTrack/GFP group and pAdTrack/TG2 group （there was no significant difference between the 2 groups）， the expression levels of Mertk protein of mouse macrophages which infected with pAdTrack/TG2/Mertk or pAdTrack/Mertk decreased obviously too （P＜0.01）， but there was no significant difference between the later 2 groups. Conclusion Gene silence adenovirus vector plasmid targeting both TG2 and Mertk synchronously is constructed successfully， and the pAdTrack/TG2/Mertk can reduce the expressions of TG2 protein and Mertk protein of mouse macrophages obviously.
Objective To investigate the differentiation of theadipose-derived adult stem cell (ADASC) induced by the recombinant adenovirus’s containing fibers derived from B-group serotype 35 (rAd5/F35)mediated human bone morphogenetic protein 7 (hBMP-7) gene and to explore a new cell sourcefor the bone tissue engineering. Methods The hBMP-7 gene wasamplified with the pcDNA1.1/AMP-hBMP-7 plasmid as a formwork. After the purification, the gene fragment was cloned into the pDC316 carrier for the recombination of the plasmid of pDC316-hBMP-7. The 293 cells were cotransfected by the skeleton plasmid of pBHG-fiber5/35 and the shuttle plasmid of pDC316-hBMP-7, and the recombinant plasmid of Ad5/F35-hBMP-7 was obtained; the recombinant plasmid of Ad5/F35enhancd green fluorescent protein(EGFP) was obtained by the similar method. The rat ADASCs were cultured and transfected by the Ad5/F35-hBMP-7plasmid and the Ad5/F35-EGFP plasmid, respectively; the remaining untransfected ADASC were used as the controls. The morphology and the growth pattern of the transfected cells were evaluated. The transcription and the expression of the transfected genes and the steogenic phenotypes such as calcium nodules and osteocalcin were evaluated by ELISA. Results The identification of PCR and enzyme cutting showed that the construction of the recombinant Ad5/F35-hBMP-7 plasmid could be confirmed. The transfection rate of the ADASC by the Ad5/F35-EGFP plasmid was determined to be greater than 90%. The hBMP-7 gene in thetransfected ADASC could express the corresponding protein, and the formation ofthe calcium nodules could be found in the induced group. The electron microscopy showed that there was a calcium element in the cytoplasm, the alkaline phosphatase result was positive, and the expression of osteocalcin was increased. Conclusion The rAd5/F35-hBMP-7 gene can promote the differentiation of the adiposederived adult stem cells to the osteoblasts in the bone tissue engineering.
Objective To explore the effects of bone marrow mesenchymal stem cells (BMSCs) transfected with adenovirus hepatocyte growth factor (Ad-HGF) on wound repair in diabetic rats. Methods BMSCs from male Wistar rats were isolated by density gradient centrifugation, cultured, and transfected with Ad-HGF. The multi pl icity of infection was 100. Diabetic models were establ ished in 20 female Wistar rats by diets in high fat and sugar plus intraperitoneal injection ofstreptozotocin (30 mg/kg). Then 2 full-thickness skin wounds (approximately 1.5 cm in diameter) were made on the dorsum. The rats were randomly divided into 4 groups (n=5 rats). After wounding, the 0.3 mL suspensions of BMSCs (group A), Ad- HGF (group B), BMSCs transfected with Ad-HGF (group C), and PBS (group D) were injected directly into the derma of wounds. The transverse diameter and longitudinal diameter of wound were measured at 21 days after treatment. At 7 days and 28 days after treatment, HE staining was performed to evaluate wound heal ing. The contents of hydroxyprol ine and advanced glycosylation end products (AGEs) in the wounds were measured by enzyme l inked immunosorbent assay and fluorospectrophotometer, respectively, at 3, 7, 14, and 28 days after treatment. Results At 21 days after treatment, the wounds almost healed in group C, and the transverse diameter and longitudinal diameter were 0 and (0.110 ± 0.024) cm, respectively. But the wounds healed partially in groups A, B, and D, and the transverse diameter and longitudinal diameter were (0.470 ± 0.051) cm and (0.590 ± 0.041) cm, (0.390 ± 0.042) cm and (0.480 ± 0.032) cm, and (0.700 ± 0.068) cm and (0.820 ± 0.068) cm, respectively. There were significant differences in wound heal ing between group C and groups A, B, and D (P lt; 0.05). The wound heal ing time of group C [(20.5 ± 1.9) days] was significantly shorter (P lt; 0.05) than those of groups A, B, and D [(28.3 ± 1.9), (25.9 ± 2.3), and (36.6 ± 5.1) days]. At 7 days, the HE staining showed that evident epidermis transportation, collagen formation, and leukocytes infiltration were observed in group C. At 28 days, the HE staining showed that the epidermis in group C was significantly thinner and more regular than those in other groups, and the decreased collagen and many small vessels were observed in group C. The content of hydroxyprol ine in group C was higher than those in groups A, B, and D at 7 days and 14 days (P lt; 0.05). The contents of AGEs in group C was lower than those in groups A, B, and D at 14 days and 28 days (P lt; 0.05). Conclusion Transplantation of BMSCs transfected with Ad-HGF can accelerate the wounds repair in diabetic rats.