To investigate the preventive effect of TGF-β1 neutral izing antibody on collagen production and adhesion formation of flexor tendon. Methods Tendon fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from 6 New Zealand rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-β along with increasing dose of TGF-β1 neutral izing antibody. Col I production was measured by enzyme-l inked immunoabsorbent assay after 3 days. Eighty-four adult New Zealand White rabbits forepaws underwent sharp transection of middle digit flexor digitorumprofundus and immediate repair. Then the rabbits were divided into three groups: the normal saline (NS group, n=36), 1.0 µg/ mL TGF-β1neutral izing antibody (1.0 µg/mL TGF-β1group, n=36) and 2.0 µg/mL TGF-β1 neutral izing antibody (2.0 µg/mL TGF-β1 group, n=12) were injected in tendon sheath respectively. Tendons were harvested at 4 and 8 weeks for biomechanics testing, histological evaluation and scanning electron microscope observation. Tendons were harvested at 1, 2, 4 and 8 weeks to determine the mRNA expression of TGF-β1 and Col I by in situ hybridization. Results ELISA exhibed that TGF-β1 enhanced Col I production and the neutral izing antibody significantly inhibited TGF-β1-induced Col I production in all 3 cell culture with a dose-dependent. At 4 and 8 weeks after operation the gl iding excursion of the tendon and the simulated active flexion in NS group were less than that of 1.0 µg/mL TGF-β1 group and 2.0 µ g/mL TGF-β1 group. There was significant difference between NS group and 1.0 µ g/mL TGF-β1 group, 2.0 µ g/mL TGF-β1 group (P lt; 0.05). The tendon anastomosis breaking strength showed no significant differences among three groups (P gt; 0.05). Scanning electron microscope and histological observation showed that collagen fibers arranged irregularly in NS group, but arranged regularly in 1.0 µ g/mL TGF-β1 group and 2.0 µ g/mL TGF-β1group at 4 and 8 weeks after operation. The in situ hybridization results revealed that TGF-β1 and Col I mRNA expression in 1.0 µ g/mL TGF-β1 group was lower than that in NS group at each time. There was significant difference between two groups (P lt; 0.05). Conclusion TGF-β1neutral izing antibody can inhibit the function of the TGF-β1 effectively and prevent adhesion formation after the flexor tendon injured and repaired.
【Abstract】 Objective To explore the preventing effects of TGF-β1 antibody (TGF-β1Ab) compounded with fibringlue (FG) on postoperative adhesions of flexor tendon. Methods Seventy-two Leghorn chickens were randomly divided into 4 groups (groups A, B, C and D), 18 chickens for each group, and the long flexor tendons of the 3rd and 4th toes in zone Ⅱ of all chickens were transversed and sutured with the 4-strand cruciate repair technique to make defect models. In group A, 0.2 mL TGF-β1 Ab was appl ied at repair site. In group B, 0.2 mL FG was appl ied at repair site. In group C, 0.2 mL TGF-β1Ab and FG was appl ied at repair site. In group D, 0.2 mL normal sodium was appl ied at repair site. At 1, 3 and 8 weeks after operation, the tendons of 6 chickens in each group were harvested for morphological and histological evaluation. Six specimens of each group were obtained for biomechanical test at 3 and 8 weeks. Results The gross observation showed that the differences ingrading of tendon adhesion were not significant among 4 groups at 1 week after operation (P gt; 0.05), but the differences were significant between groups A, B, D and group C at 3 and 8 weeks after operation (P lt; 0.05). Histological observation showed that collagen fibers arranged irregularly in groups A, B and D, but arranged regularly in group C at 3 and 8 weeks after operation. At 3 weeks after operation the gl iding excursion ratio of the tendon in groups A, B, C and D were 0.45 ± 0.05, 0.40 ± 0.10, 0.79 ± 0.09 and 0.25 ± 0.07 respectively ; the simulated active flexion ratio were 0.61 ± 0.02, 0.67 ± 0.03, 0.91 ± 0.03 and 0.53 ± 0.04 respectively; the work of flexion were(18.00 ± 0.77), (17.80 ± 1.13), (27.60 ± 1.73) and (15.60 ± 1.27)?/N respectively. There were significant differences between group C and other three groups (P lt; 0.05). The tendon anastomosis breaking strengthwere (14.2 ± 1.9), (15.2 ± 2.2), (16.0 ± 2.2) and (14.7 ± 2.7) N, showing no significant differences among 4 groups (P gt; 0.05).At 8 weeks after operation, the gl iding excursion ratio of the tendon in groups A, B, C and D were 0.45 ± 0.07, 0.43 ± 0.08, 0.80 ± 0.09 and 0.29 ± 0.05 respectively; the simulated active flexion ratio were 0.61 ± 0.02, 0.63 ± 0.03, 0.92 ± 0.03 and 0.53 ± 0.03 respectively, the work of flexion were (18.30 ± 0.84), (18.60 ± 0.80), (27.90 ± 1.24) and (15.30 ± 0.75) ?/N respectively. There were significant differences between group C and other three groups (P lt; 0.05). The tendon anastomosis breaking strength were(51.9 ± 3.0), (51.4 ± 1.4), (53.3 ± 1.3) and (52.3 ± 2.2) N, showing no significant differences among 4 groups (P gt; 0.05). Conclusion TGF- β1Ab compounded with FG could significantly prohibit the formation of fibrous adhesions without interfering with the heal ing process.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors’ il iac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were establ ished. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-β3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-β3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were appl ied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P lt; 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P lt; 0.05). The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
目的：对TGF-β1的G-800A，C-509T，T869C和G915C多态性位点与IgA肾病及其临床表现之间的相关性进行了分析。方法：纳入119例经肾活检证实为IgA肾病的患者和116例健康正常成人作为对照组。采用多聚酶链式反应-限制片段长度多态性分析和多聚酶链式反应-扩增受阻突变体系方法测定TGF-β1的G-800A，C-509T，T869C和G915C多态性位点的基因型，分析其与IgA肾病发病以及临床表现的相关性。结果：研究对象中G-800A和G+915C这两个位点没有多态性。IgAN患者C-509T位点的等位基因型分布与正常对照具有显著性差异（Ｐ＜0.05），且IgAN患者TT纯合子的频率显著高于对照组（33% vs 20%,Ｐ=0.02）。C869T位点等位基因的分布无显著性差异。C-509T和C869T位点等位基因分布和患者临床表现无明显的相关性。结论：TGF-β1基因C-509T位点的T等位基因携带可能与IgA肾病的发病易感性具有相关性。
【Abstract】Objective To investigate the expression of tumor growth tactor β1 (TGFβ1) and p27 in gallbladder carcinoma and their relation to the development of the carcinoma. Methods The expression of TGF-β1 and p27 in 36 cases of gallbladder carcinoma was detected by SP immunohistochemical staining. Twenty cases of chronic cholecystitis were collected as control. Results The positive rate of TGF-β1 （63.9%） was higher than that of the control （10.0%），P＜0.05， and the positive rate of p27 (47.2%) was lower than that of the control 100%(P＜0.05). The positive rate of TGF-β1 was significantly higher in metastasis or Nevin Ⅳ～Ⅴ stage cases than that of non-metastasis or Nevin Ⅰ～Ⅲ stage cases 33.3% (P＜0.05). The positive rate of p27 was statistically higher in moderate and highly differentiation (60.9%), nonmetastasis (75.0%) or Nevin’s Ⅰ～Ⅲ stage (75.0%) cases than those of poor differentiation (23.0%), metastasis (33.3%) and Nevin Ⅳ～Ⅴ stage (33.3%) cases (P＜0.05). The expression of p27 was negatively correlated with that of TGF-β1(r=-0.4473，P＜0.05). There was significant difference in survival time between patients with TGF-β1 positive and TGF-β1 negative（P＜0.05）. The difference was also found between patients with p27 positive and p27 negative. Conclusion The upregulation of TGF-β1 and downregulation of p27 in gallbladder carcinoma indicates the imbalance of TGF-β1/p27 system, which may play a role in the carcinogenesis and predict the malignant behaviors of the carcinoma.
【Abstract】 Objective To summarize the recent progress in related research on transforming growth factor β1 （TGF-β1）/Smad3 signal transduction pathway and post-traumatic scar formation. Methods Recent related literature at home and abroad on TGF-β1/Smad3 signal transduction pathway and post-traumatic scar formation was reviewed and summarized. Results TGF-β1 is an important influence factor of fibrotic diseases, and it plays biological effects by TGF-β1/Smad3 signal transduction pathway. The pathway is regulated by many factors and has crosstalk with other signal pathways at cellular and molecular levels. The pathway is involved in the early post-traumatic inflammatory response, wound healing, and late pathological scar formation. Intervening the transduction pathway at the molecular level can influence the process of fibrosis and extracellular matrix deposition. Conclusion TGF-β1/Smad3 signal transduction pathway is an important way to affect post-traumatic scar formation and extracellular matrix deposition. The further study on the pathway will provide a theoretical basis for promotion of wound healing, as well as prevention and treatment of pathological scar formation.
Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated frombone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs wereharvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected bylaser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.