Objective Targeted adenoviral gene delivery from peripheral nerves was used to integrally analyse the characterization and time course of LacZ gene (AdLacZ) retrograde transfer to spinal cord and transgene product anterograde labeling ofperipheral nerve. Methods Recombinant replication-defective adenovirus containing AdLacZ was administrated to the cut proximal stumps of median and tibial nerves in Wister rats. Then the transected nerve was repaired with 10-0 nylon sutures. At different time point postinfection the spinal cords of C5 to T1 attached with DRGs and brachial plexuses, or L2 to L6 attached with DRGs and lumbosacralplexuses were removed. The removed spinal cord and DRGs were cut into 50 μm serialcoronal sections and processed for X-gal staining and immunohistochemical staining. The whole specimens of brachial or lumbosacral plexuses attaching with theirperipheral nerves were processed for X-gal staining. The number of X-gal stained neurons was counted and the initial detected time of retrograde labeling, peaktime and persisting period of gene expression in DRG sensory neurons, spinal cord motor neurons and peripheral nerves were studied. Results The gene transfer was specifically targeted to the particular segments of spinal cord andDRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. Within the same nerve models, the initial detected time of gene expression was earliest in DRG neurons, then in the motor neurons and latest in peripheral nerves. The persisting duration of β-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time of β-gal staining in median nerve models was earlier in mediannerve models compared with that in the tibial nerve models. Although the initial detected time and the beginning of peak duration of β-gal staining were not same, the decreasing time of β-gal staining in motor and sensory neurons of thetwo nerve models were started at about the same day 8 post-infection. The labeled neurons were more in tibial nerve-models than that in median nerve models. Within the same models, the labeled sensory neurons of DRGs were morethan labeled motor neurons of ventral horn. The β-gal staining was tenser in median nerves than that in tibial nerves. However the persisting time of β-gal staining was longer in tibial nerve models. Conclusion The b gene expression in neurons and PNS renders this system particularly attractive for neuroanatomical tracing studies. Furthermore this gene delivery method allowing specific targeting of motor and sensory neurons without damaging the spinal cord might offer potentialities for the gene therapy of peripheral nerve injury.
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
【Abstract】 Objective To detect the expression of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3)in cell death induced by nutrition deprivation in nucleus pulposus cells so as to further understand the mechanism of deathin nucleus pulposus cells. Methods Two adult Sprague Dawley rats, male or female, weighing 150-200 g, were involvedin this experiment. The cells isolated from rat caudal disc were cultured under the condition of L-DMEM culture media,10%FBS, and 21%O2 (control group) and under the condition of DMEM-free glucose culture media, no serum, and 1% O2(experimental group). The expressions of BNIP3 gene and protein were detected by real-time fluorescent quantitative PCR,immunofluorescence staining, and Western blot. The cell apoptosis rate and mitochondrial membrane potential were measuredby flow cytometry at 24, 48, and 72 hours after culture. Results The expression of BNIP3 decreased in the control group;the expressions of BNIP3 showed an increasing tendency with time in the experimental group, and BNIP3 combined withmitochondria. Significant differences were observed in the expressions of BNIP3 gene and protein between 2 groups at the othertime (P lt; 0.05) except that no significant difference was observed in the expression of BNIP3 gene at 24 hours (P gt; 0.05). Thecell apoptosis rate and mitochondrial membrane potential were significantly lower in the experimental group than those in thecontrol group (P lt; 0.05). Conclusion Upregulation of BNIP3 and translocation to mitochondria may be involved in nucleuspulposus cell death in nutrition deprivation.
【摘要】 目的 觀察已構建的含胸苷激酶（TK）自殺基因的重組腺病毒（ADV-TK）對肝癌細胞的體外殺傷作用和對肝癌裸鼠移植瘤的治療效果。 方法 將ADV-TK體外感染人肝癌細胞株SMMC-7721，噻唑藍（MTT）法檢測受感染的SMMC-7721細胞被不同濃度更昔洛韋（GCV）作用后的細胞存活率情況。構建肝癌SMMC-7721裸鼠移植瘤模型，觀察腫瘤注射重組腺病毒ADV-TK結合GCV治療移植瘤的變化。 結果 相同滴度的重組腺病毒與不同濃度的GCV作用于肝癌細胞株SMMC-7721后，MTT法檢測到細胞的存活率隨著GCV濃度的增加而不斷降低。動物實驗中ADV-TK治療組腫瘤體積明顯小于對照組（ADV-null及NS）（Plt;0.01）。 結論 重組腺病毒ADV-TK對肝癌SMMC-7721細胞的體外增殖和裸鼠體內的移植瘤生長均有明顯的抑制作用。【Abstract】 Objective To explore the inhibitory effect of recombinant adenovirus containing TK gene (ADV-TK) on transfected human liver cancer cells SMMC-7721 in vitro and murine transplanted hepatocarcinoma in vivo. Methods SMMC-7721 cells transfected with ADV-TK were exposed to medium with GCV. The cell viability was measured by MTT assays. In the established model of SMMC-7721 human liver cancer, nude mice underwent intratumoral injection with 1 109 pfu ADV-TK, the control vector (ADV-null) or normal saline (NS) and again 7 days later, twice for all. GCV was given at a dose of l00 mg/(kg?d) on the following day of injection for 10 days. The tumor inhibitory effect was observed by measuring the tumor sizes. Results After transfected by ADV-TK in vitro, and combined with GCV, the cell growth of SMMC-7721 cell were significantly suppressed. The result of in vivo assay showed that tumor volumes in treatment group were apparently smaller than that in the control group (Plt;0.01). Conclusion Recombinant adenovirus combined with GCV shows a significant inhibitory effect on SMMC-7721 cells in vitro and murine transplanted hepatocarcinoma in vivo.
ObjectiveTo construct a transgenic cell sheet of cartilage-derived morphogenetic protein 1 (CDMP-1) by adenovirus vector in vitro and to identify its biological activity.
MethodsThe bone mesenchymal stem cells (BMSCs) were isolated from bone marrow of 1-month-old rabbit, and cultured in vitro. The 3rd-6th generation of BMSCs were used for experiment. The experiment was divided into 3 groups:BMSCs transfected by adenovirus (Ad)-cytomegalovirus (CMV)-human CDMP1 (hCDMP1)-internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP) in group A, BMSCs transfected by Ad-CMV-EGFP in group B, and untransfected BMSCs in group C. The expression of green fluorescence was observed in 3 groups under fluorescent inverted microscope. MTT assay was used to detect the proliferation of the cells. The cell sheet was obtained by means of temperature-responsive culture dish for 14 days. The morphological and HE staining observations of the cell sheet were carried out. RT-PCR and Western blot were used to detect the expressions of hCDMP1 and collagen type II at gene and protein levels, while alcian blue staining was used to detect the expression of glycosaminoglycans (GAG).
ResultsBright green fluorescence was observed in transfected cells at 72 hours under fluorescent inverted microscope, and the transfection efficiency was up to 90%. MTT assay showed approximate S-shaped growth curves in 3 groups, showing no significant difference in the absorbance (A) value among 3 groups within 9 days (P>0.05). The three-dimensional cell sheets were successfully harvested in vitro. The RT-PCR and Western blot showed that there were positive expressions of hCDMP1 and collagen type II in group A and negative expression in other 2 groups. HE staining and alcian blue staining showed that there were rich fibrous tissues, mass extracellular matrix, and dark blue metachromatic granules in group A, but there was less fibrous tissues and no specific blue metachromatic granules in other 2 groups; and the positive expression area was significantly lower and gray scale of GAG was significantly higher in group A than that in groups B and C (P<0.05).
ConclusionA transgenic cell sheet of exogenous recombinant hCDMP1 by adenovirus vector can express collagen type II and GAG, so it has chondrogenic capacity. This technology that overcomes limitations in traditional tissue engineering, such as low cell-attachment efficiency and inflammatory reaction, may be a new tissue engineering approach for hard tissue reconstruction and is hopeful to build a large density of tissue engineered cartilage.
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.
Objective To construct a replicationdefective recombinant adinovirus including the target gene human bone morphogenetic protein 4(fragment hBMP-4). Methods The hBMP-4 gene fragment was cut down from pCS2(+)/hBMP-4, cloned into the eukaryotic expressive vector pcDNA 3.1(+), then subcloned into pShuttle-CMV and transformed into the competent E. coli BJ5183/p by electroporation. The resulting recombinant plasmid pAdE/hBMP-4 was transformed into the packaging of thecell lines HEK293 to produce the replication-defective recombinant adenovirusescontaining the hBMP-4 gene. These replication-defective recombinant adinoviruses were transfected into HEK293 and HeLa cells. Then, total RNA and total protein were detected by RT-PCR and the Western-blot assay. Results The pAdE/hBMP-4 was confirmed by the restrictional endonuclease digestion. In HEK293 and HeLa cells, the specific transcription of the hBMP-4 gene was confirmed by RT-PCR, and the expression of the hBMP-4 protein was confirmed by theWestern-blot assay. Conclusion The replication-defective recombinant adinovirus expression vector containing the hBMP-4 gene can be constructed and expressed successfully, which has laid a foundation for the further research on the genetherapy of hBMP-4.
Objective To observe the tissue engineered bonefabricated with the cultured mesenchymal stem cells (MSCs) by the green fluorescent protein (GFP) gene transfer. Methods The recombinant Adeno-XTM-GFP expression vector was purified after being packed and proliferated by the HEK293 cells, and then it was used to infect the rabbit’s MSCs directly afer the virus titer was assayed. The cell morphological changes were observed under the inverted phase contrast microscope, and the expression of GFP was observed under the fluorescence microscope to confirm success of the labeling of MSCs.The GPFlabeled MSCs and the pure MSCs were cultured together in the conventional osteogenic supplements for 3 weeks, and then they were seeded onto the compound scaffold of the calcium phosphate cement (CPC) and the fibrin glue (FG) to form a new kind of the tissue engineered bone. It was implanted into the donator rabbit subcutaneously to be used as the experimental group; in contrast, the pure compound scaffold of the CPC-FG without any MSCs was implanted in the same rabbit as a control. The alkline phosphatase (ALP) activity assay was performed respectively at 1, 2 and 3 weeks after operation. GFP was observed under the laserconfocal microscope at 4 weeks after operation, and the formed new bone was harvested at 4 weeks and evaluated by the Masson staining, the immunohistochemistry staining of osteocalcin (OC) and collagen typeⅠ.Results The virus titer was 3×108pfu/ml after proliferation and purification. Expresstionof GFP was confirmed 96 h after MSCs were infected by the Adeno-XTM-GFP expression vector and the infection rate was proximally 50%-70%. In contrast to MSCs, division and proliferation of the GPF-labeled MSCs were not significantly different. The ALP activity in the experimental group (12.546±1.091, 16.567±0.659, 20.443±0.706) was significantly higher than that in the control group (0.453±0.113, 0.243±0.018, 0.308±0.056), respectively at 1, 2 and 3 weeks after operation (Plt;0.05). The tissue engineered bone formed at 4 weeks. There were newly-formed trabeculae around the pore of the compound scaffold, and theimmunohistochemistry staining of OC and collagen typeⅠ were positive. The laser confocal microscope revealed that the GFP-labeled cells existed in many newlyformed tissues，and the compound scaffold of CPC-FG was partly degraded. Conclusion The engineered bone is similar to the spongy bone and the composed cells originate from the cultured MSCs, all of which can be confirmed by the GFP gene transfer technique.
Objective
To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor α (TNF-α).
Methods
The primer of small interfering RNA (siRNA) coding sequence of silent TNF-α was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 × 109 PFU/mL), and Ad5-TNF-α-siRNA-CMVeGFP (1 × 109 PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-α was detected by Western blot at 12 weeks after operation.
Results
The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-α-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A lt; group D lt; group B lt; group C, P lt; 0.05). Western blot showed that the TNF-α expression levels were 0.235 ± 0.022, 0.561 ± 0.031, 0.731 ± 0.037, and 0.329 ± 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P lt; 0.05).
Conclusion
The recombinant adenovirus for silencing TNF-α is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-α expression in the tissues around prosthesis in mice.
