【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.
【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.
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.
ObjectiveTo investigate the regulatory mechanism of transforming growth factor-β1 (TGF-β1) in different types of mitral valvular disease (MVD) with atrial fibrillation (AF). MethodsFrom August 2011 to August 2012, patients with moderate to severe MVD accompanied by AF who required mitral valve replacement at the Department of Cardiovascular Surgery, West China Hospital, Sichuan University, were included. Based on echocardiographic results, patients were divided into two groups: a mitral regurgitation (MR) with AF (MR-AF) group and a mitral stenosis (MS) with AF (MS-AF) group. Left atrial tissue samples were collected during surgery. Techniques such as enzyme-linked immunosorbent assay, real-time fluorescence quantitative polymerase chain reaction, immunohistochemistry, and Western blotting were used to detect key molecules in the TGF-β1/JNK pathway. ResultsSixteen patients were enrolled. There were 8 patients in the MR-AF group, including 5 males and 3 females, with an average age of (41.38±11.19) years; and 8 patients in the MS-AF group, including 6 males and 2 females, with an average age of (43.12±5.30) years. The left atrial volume load was higher in MR-AF patients, while the left atrial pressure load was higher in MS-AF patients. In MS-AF patients, the relative expression levels of MAPK9, JUN, CASP3, BAX, and BCL2 mRNA in left atrial tissues were significantly upregulated. The serum TGF-β1 protein level and the relative expression levels of p-JNK, p-c-Jun, and Caspase-3 proteins in the left atrial tissues of the MR-AF group were higher. Myocardial cell damage was more severe in the MS-AF group, and the protein expression level of Bcl-2 was higher. ConclusionDifferent MVD have distinct hemodynamic characteristics. The myocardium of the left atrium in MR-AF patients is more prone to apoptosis, possibly through the activation of the TGF-β1/JNK signaling pathway.
Objective To determine the efficacy of D980-nm laser in dissolving fat and renewing skin, and to explore the clinical application of D980-nm laser in reconstruction of photodamaged skin. Methods Eighteen 12-14 month-old male Sprague-Dawley rats, weighing 400-450 g, were randomly divided into 3 groups (n=6). The rat skin at the left side was exposed to D980-nm laser irradiation at a density of 20 J/cm2, a power of 8 W, a pulse width of 20 ms, and a pulse frequency of 40 Hz for 1 time (group A), 2 times of 5-minute interval (group B), and 3 times of 5-minute interval (group C) as a treatment course, for 4 treatment courses with an interval of 1 week; the other side of the skin was not treated as the control groups (groups A1, B1, and C1, respectively). After 8 weeks, the skin was harvested for HE staining and immunohistochemical staining to observe the structure changes of skin, to measure the dermal thickness, to count the number of fibroblasts, and detect the expressions of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor (bFGF). Results Compared with groups A1, B1, and C1, the skin structure was significantly improved in groups A, B, and C. After D980-nm laser irradiation, the number of fat cells decreased; local angiogenesis was observed; the total number of fibroblasts and fibers increased; the collagen fiber had large diameter, and arranged closely and regularly; the dermal thickness and the number of the fibroblasts increased; and the expressions of TGF-β1 and bFGF were significantly enhanced, showing significant differences (P<0.05). With increased D980-nm laser irradiation times, the above indexes increased, showing significant differences between group C and groups A, B (P<0.05). Conclusion D980-nm laser treatment has lipolytic and tender effect on the skin, and the frequency of the treatment is an important factor in skin renewal.
Objective To access the protective effects of glucosamine hydrochloride capsules (OTL) on articular cartilage in osteoarthritis of rabbit. Methods Thirty-six New Zealand white rabbits were divided randomly into three groups (n=12): sham group (group A), anterior cruciate l igament transection (ACLT)/normal sal ine group (group B), and ACLT/ OTL group (group C). Rabbits in groups B, C received ACLT on the right knee. Rabbits in group A were not given ACLT ascontrol. Group C received a daily administration of OTL at a dose of 150 mg/kg of body weight for 12 weeks; in contrast, group B received normal sal ine at the same dose. All rabbits were sacrificed after 12 weeks. The right femoral condyle were removed and observed at pathologic changes with HE staining and graded by Mankin’s scale, the expression level of transforming growth factor β1 (TGF-β1) and interleukin 1β (IL-1β) were detected by immunohistochemical staining. Results All rabbits survived at the end of experiment and incision healed well. The gross observation showed that joint synovia increased and articular surface was smooth and integrity in group A; that ulcer was observed on the articular surface of group B; and that articular surface was smooth and integrity in group C. There were sigificant differences in articular cartilage scores between 3 groups (P lt; 0.05). The histological observation showed that the articular cartilage had normal structure and the cells arranged regularly in group A; that the articular cartilage became thin and the cells arranged irregularly in group B; and that the cells arranged with a clear layer and had regular shape in group C. The Mankin scores were 1.04 ± 0.13, 7.97 ± 0.12, and 2.81 ± 0.36 in groups A, B, and C, respectively; showing significant difference between 3 groups (P lt; 0.05). The result of immunohistochemistry showed that the expressions of TGF-β1 were 50.62 ± 1.51, 24.81 ± 1.28, and 41.57 ± 1.69 and the expressions of IL-1β were 13.12 ± 1.21, 62.53 ±2.37, and 30.67 ± 1.28; showing significant differences between 3 groups (P lt; 0.05). Conclusion A daily administration ofOTL at a dose of 150 mg/kg for 12 weeks can partially decrease the expression levels of IL-1β and increase the expression levels of TGF-β1, which delays the development of osteoarthritis.
PURPOSE: To investigate the influence of transforming growth factor-beta (TGF-beta;) on endotoxin-induced uveitis (EIU). METHODS: TGF- beta; was abstracted from humam platelets using Bio-gel P-60 chromatography. It was introduced either topically(drops) or intraperitoneally into the SD rats after footpad injection of lipopolysaccbaride(LPS). The inflammation in the anterior uvea was clinically evaluated with slitlamp every day. RESULTS :TGF-beta; obtained from Biogel P-60 chromatography displayed single band on SDS-PAGE,showing a molecular weight of 12 500 EIU occurs significantly earlier and more severe in the rats which only received LPS injection than in the TGF-beta; treated rats. The duration of the inflammation was also much longer in the untreated rats than in the treated rats (P＜0. 001). CONCLUSION: TGF-beta; purified from human platelets may partly prevent the development of EIU and effectively reduce the severity of the inflammation induced by LPS injection. （Chin J Ocul Fundus Dis,1996,12: 101-104)
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001). ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.