Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
Abstract： Objectives To determine the atrial expression of the collagen Ⅰ, collagen Ⅲ and the transforming growth factorbeta 1 (TGF-β1) in patients with rheumatic heart disease (RHD) and permanent atrial fibrillation(PAF) and to investigate the relationship between the extent of atrial fibrosis and the effectiveness of radiofrequency maze procedure in patients with RHD and PAF. Methods A total of 40 patients with RHD and PAF (≥6 months) who underwent a radiofrequency maze procedure with concomitant valvular surgery were collected for the experimental group. We acquired 100 mg of the left auricle tissue in each patient and followed up these patients after 3, 6 months of [CM(158mm]surgery. Then we assigned these patients to nonAF group and persistent AF group according to the results of the 6month followup. Another 10 patients with RHD and sinus rhythm(SR) who underwent valvular surgery alone were assigned to SR group and their left auricle tissue was also obtained. In order to determine the extent of atrial fibrosis, we observed the amount of collagen volume fraction Ⅰ，Ⅲ(CVF-Ⅰ,CVF-Ⅲ) by semiquantitative analysis with picrosirius red staining method. Using the β actin protein as the endogenous reference gene, we detected the expressions of TGF-β1 mRNA by semiquantitative reverse transcriptionpolymerase chain reaction(RT-PCR) technique. Results Each group has the same clinical baseline. At 6month follow-up, 28 among the 40 patients were categorized into the nonAF group and 12 into the AF group. (1) Patients in the nonAF group and the AF group had higher mRNA expressions of TGF-β1, CVF-Ⅰ and CVF-Ⅰ/CVF-Ⅲ compared with the SR group (F=6.487, P=0.003; F=3.711, P=0.032; F=3.697, P=0.032). The AF group had higher mRNA expressions of TGF-β1, CVF-Ⅰ and CVF-Ⅰ/CVF-Ⅲ than the nonAF group (t=4.372, P=0.043; t=4.603, P=0.038; t=4.776, P=0.035). But the CVFⅢ had no significant differences among the three groups (P＞0.05). (2) The patients whose left atrial function recovered after Maze procedure had lower mRNA expression than those patients whose left atrial function did not recover in the nonAF group (t=5.570, P=0.027). (3) The TGF-β1 mRNA expression has a positive correlation with both the contents of CVF-Ⅰ and left atrial diameter (r=0.786, Plt;0.05; r=0.858, Plt;0.05). Multiple logistic regression analysis revealed that the mRNA expression levels of TGF-β1, CVF-Ⅰ and left atrial diameter were independently associated with the postoperative persistence of atrial fibrillation. Conclusion The extent of atrial fibrosis in patients with RHD and PAF may be related to the sinus rhythm restoration and maintenance after AF surgical radiofrequency ablation and the resumption of atrial function.
Objective To study the inhibitory effects of curcumin on bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage and explore its possible mechanism.Methods 96 male SD rats were randomly divided into a normal control group，a fibrosis model group，a fibrosis model treated with prednisone group and a fibrosis model treated with curcumin group.Pulmonary fibrosis were induced by instilled bleomycin through tracheal.From day 15 after bleomycin administration，the curcumin group and prednisone group were given curcumin（300 mg/kg） or prednisone（5 mg/kg） per day by intragastric administration，respectively.The normal control group and fibrosis model group were given 1% sodium carboxymethyl cellulose（10 mL/kg） as control.Six rats of each group were randomly sacrificed on day 21，28，42 and 56 after bleomycin administration，respectively.The histological changes of the lung were evaluated by HE and Masson’s trichrome staining.Lung expressions of transforming growth factor-β1（TGF-β1） and hydroxyproline were assessed by immuno-histochemistry and digestion method，respectively.Results Pulmonary fibrosis and hydroxyproline level in the curcumin group were significantly reduced as compared with those in the model group on day 42 and 56.The expession of TGF-β1 in the curcumin group was significantly lower than that in the model group on day 28，42 and 56，and was not significantly different from the normal group on day 56.Conclusion Curcumin could alleviate bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage by inhibiting the expressions of TGF-β1.
Objective To investigate the expression of transforminggrowth factor β1(TGF-β1) and insulin-like growth factorⅠ(IGF-Ⅰ)in new bone after low frequency micromovement. Methods Fifteen female sheep from Shandong province were involved in the study and their bilateral tibias transversely osteotomized in the middle shafts with a defect of 2 mm.The hind limbs were fixed with unilateral external fixators connected to a controlled micromovement device. Ten days after osteotomy, one hind limb of each sheep randomlywas selected to perform micromovement at an amplitude of 0.25 mm and a frequency of 1 Hertz, 30 min a day for 4 weeks ( micromovement group). The other hindlimb served as the control group. Five sheep were sacrificed at 3,4 and 6 weeks after osteotomy, respectively, and specimens were harvested for detecting the expression of TGF-β1 and IGF-Ⅰby immunohistochemistry and RT-PCR. Results Immunohistochemistry: In the third postoperative week in the micromovement group, the expression of TGF-β1 was detected in different areas of new chondrocytes at the margin of callus, mainly in proliferating area, and IGF-Ⅰexpressed in osteoblasts at the margin of endochondral ossification area, calcified and mature chondrocytes and osteocytes. There was seldom expression ofIGF-Ⅰ and little expression of TGF-β1 in the corresponding area in the control one. In the 4th postoperative week in the micromovement group, theexpression of TGF-β1 diminished gradually with the mature of new bone and be located in extracellular matrix and osteoblasts around ossified areas; The expression ofIGF-Ⅰ reached the peak and be located mainly in osteoblasts of new bone surface, maturing osteocytes and calcifing osteoid. But there was little expression of them in the control group. In the sixth postoperative week in the micromovement group, there was a little expression of IGF-Ⅰ expression but little expression of TGF-β1; there was nearly no expression of them in the control group. In the micromovement group, the absorbance values of TGF-β1 at 3 and 4 weeksand of IGF-Ⅰat 3, 4 and 6 weeks were significantlyhigher than those in control group（P＜0.05）. RTPCR: In the third and fourth postoperative weeks in the micromovement group, there was higher expression of mRNA of TGF-β1 and TGF-I than those in control group; in the sixth postoperative week, the expression diminished gradually, but was higher than that in control group. The absorbance values of TGF-β1 at 3 and 4 weeks and IGF-Ⅰat 3, 4 and 6weeks were significantly higher than those of control group（P＜0.05）. Conclusion Low frequency and controlled micromovement in the early stage of the fracture healing can promote the expression of TGF-β1 and IGF-Ⅰ.They worked together to regulate the process of the endochondral ossification, while in the late stage the differentiation of osteocytes and mineralization of osteoid were regulated mainly by IGF-Ⅰ, which played an important role in regulating the cell biological behavior during micromovement.
目的：研究转化生长因子β1（TGF-β1）促进Tenon囊成纤维细胞的增殖作用及核心蛋白多糖（decorin）对TGF-β1促Tenon囊成纤维细胞增殖的影响。方法：（1）取青光眼患者的Tenon囊组织，采用组织块贴壁培养法进行原代培养细胞培养，细胞经过冷冻与复苏后，观察细胞形态结构与生物学特性。（2）用不同浓度(0.01、0.1、1、5、10ng/mL)TGF-β1作用于Tenon囊成纤维细胞48 h。（3）终浓度为5 ng/mL的TGF-β1 和不同浓度(0.01、0.1、1、2、5μg/mL)的decorin共同作用于Tenon囊成纤维细胞48h。MTT比色法检测Tenon囊成纤维细胞增殖。结果：（1）体外成功培养Tenon囊成纤维细胞，细胞生长活跃，形状稳定，细胞经过冷冻与复苏后，仍能保持其原来的细胞形态结构与稳定的生物学特性。（2）1～10ng/mL TGF-β1能促进Tenon囊成纤维细胞的增殖，并与浓度成正比。（3）2～5μg/mL decorin 能抑制TGF-β1促Tenon囊成纤维细胞增殖作用，其抑制作用与浓度成正比。结论： TGF-β1可刺激Tenon囊成纤维细胞增殖，Decorin能抑制TGF-β1促进细胞增殖的作用，其机理可能通过与TGF-β1结合抑制青光眼滤过手术后瘢痕形成。
ObjectiveTo study the serum transforming growth factorβ1 (TGF-β1) and interleukin-23 (IL-23) expression in the patients with chronic gastric ulcer or gastric cancer, and to investigate the clinical value of TGF-β1 and IL-23 on the prevention and treatment of gastric cancer. MethodsThe serum levels of TGF-β1 and IL-23 in cancer group (83 cases), gastric ulcer group (184 cases), and control group (58 cases) were detected by using ELISA assay method. The difference of serum TGF-β1 and IL-23 levels in patients with gastric cancer with different pathological parameters were compared. ResultsThe serum levels of TGF-β1〔(15.96±3.92) ng/mL〕and IL-23〔(645.25±234.18) ng/mL〕in gastric cancer group were higher than those of the gastric ulcer group〔(10.10±3.58) ng/mL, (496.10±108.32) ng/mL〕and normal control group〔(9.87±2.86) ng/mL, (372.75±89.27) ng/mL〕, the difference were statistically significant (P < 0.05). The levels of serum TGF-β1 in gastric cancer patients of stageⅠ-Ⅱ, ⅢandⅣwere successively increased, and the differences were statistically significant (P < 0.05). The levels of serum TGF-β1 in poorly differentiated gastric cancer or with lymph node metastasis patients were higher than those in high-middle differentiation or without lymph node metastasis patients, the difference were statistically significant (P < 0.05). There were no significant difference in the levels of serum TGF-β1 between different tumor diameter and different location (P > 0.05). The level of serum IL-23 in patients with stageⅠ-Ⅱwas higher than that in stageⅢandⅣ, the difference was statistically significant (P < 0.05). Ther were no significant difference in serum IL-23 levels between the different degree of differentiation, lymph node metastasis or not, different tumor diameter and different location of the tumor (P > 0.05). ConclusionTGF-β1 and IL-23 have important reference value in judging the stage and malignancy degree of gastric cancer.
Objective To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-t ransforming growth factor-β1 (TGF-β1) gene and to evaluate the feasibility of selfinduction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further “gene enhanced tissue engineering” research. Methods The rabbit MSCs was transfected with the reconstructed PGL3-TGF-β1gene by the Liposo mesMethod, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evalua ted by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-β1 and collagen Ⅱ were examined at 2 and 7 days of the cell culture afte r transfe ction with PGL3-TGF-β1gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was perfor med with the software of the SPSS 11.0, compared with the control group and the blank group. Results Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-β1gene by the Liposomes Method achie ved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (Plt;0.01). Tested by MTT, the living acti vity of the transfected cells was proved to be significantly decreased (Plt;0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF-β1 positive particles were detected in the experimental group，but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the ttest (Plt;0.01). By the immunohistochemistry me thod (SABC) to study collagen Ⅱ, there were more positive particles in the transfected cells in t he experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (Plt;0.01). Conclusion Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-β1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfecte d BMS cells with PGL3-TGF-β1 gene can express and excrete TGF-β1when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.
Objective To investigate the expressions of transforming growth factor (TGF) -α and -β1 after 90% portal branch ligation (PBL) in rats. Methods Ninety-six SD rats were randomly divided into sham operation group and portal vein branches ligation group. The weight of both ligated and unligated lobes of liver were measured on 0.5, 1, 3, 5, 7, 14, 21, and 28 d after operation. The morphological changes of the unligated liver lobes were observed by microscope. The expressions of proliferating cell nuclear antigen (PCNA), TGF-α, and TGF-β1 of the unligated liver lobes were detected by immunohistochemistry. Results After the ligation of 90% portal vein branches, hepatic lobe at the ligated side diminished progressively after ligation, whereas the lobes of the unligated side underwent compensatory regeneration. The ratio of unligated lobes weight to the whole liver increased slowly within 1 d, speeded up significantly during 1-5 d period, increased slowly on 5 d, and reached plateau phase on 7 d after operation. The expressions of PCNA protein markedly increased within 0.5-3 d (Plt;0.01), which reached the peak on 5 d and decreased slightly on 7 d after operation, but still higher than sham operation group level, and then gradually returned to the level of sham operation group lately. The expressions of TGF-α and TGF-β1 in the unligated liver lobes markedly increased on 0.5 d, and reached the peak on 3 d and 1 d respectively， and then gradually returned to the level of sham operation group in 7-28 d after operation. Conclusion Ligation of 90% portal branches can induce active regeneration of unligated liver lobes in rats, whose initiation and proliferation are involved in the expressions of TGF-α and TGF-β1 protein.
Objective To investigate the expression of fibroblast activation protein( FAP) in mouse pulmonary fibrosis and the relationship between FAP and trarisforming growth factor β1 ( TGF-β1 ) .Methods The mouse model with pulmonary fibrosis was established by intratracheally instillation of bleomycin. The expressions of FAP, α-smooth muscle action( α-SMA) , and TGF-β1 in lung tissues were detected with immunohistochemical technique. Results There was no expression of FAP in the normal lung tissue. In the pulmonary fibrosis mice, FAP was found in the fibroblasts existed in bronchiolar adventitia or peribronchiolar ( and perivascular) connective tissue. In the fibroblast foci, the localizations of α-SMA, TGF-β1 , and FAP were similar, but the expression of FAP was more extensive than that of α-SMA and ber than that ofTGF-β1 . The degree of lung fibrosis was bly correlated with FAP, α-SMA, and TGF-β1 ( r = 0. 795,0. 766,0. 628; P lt; 0. 01) . A significantly positive correlation was also observed between TGF-β1 and FAP( r =0. 706, P lt; 0. 01) . Conclusions FAP is especially expressed in the fibroblast foci in pulmonaryfibrosis and bly correlated with the severity of pulmonary fibrosis. FAP and TGF-β1 possibly play a synergistic role in the progression of pulmonary fibrosis.