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.
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 the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
Objective To investigate the effects of bursopentin ( BP5) on expression of extracellular matrix in human lung fibroblasts ( HLFs) and its mechanism.Methods HLFs were cultured in vitro and divided into five groups. The cells in the control group were cultured in DMEMwithout TGF-β1 or BP5. The cells in TGB-β1 treatment group were cultured in DMEMcontaining 5 μg/L TGF-β1 . While in three TGF-β1 + BP5 treatment groups, the cells were cultured in DMEM containing 5 μg/L TGF-β1 and simultaneously intervened with BP5 at three different concentrations ( 2. 5 μg/mL, 5 μg/mL, and 10 μg/mL respectively) . The expression of α-SMA was detected using a fluorescent-labeling strategy. The expressions of Collagen-Ⅰ, p-Smad2/3, p-Smad3, and Smad7 proteins were measured by Western blot. Results The cells in the TGF-β1 treatment group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In the TGF-β1 treatment group, the expressions of collagen-Ⅰ( 1. 402 ±0. 158 vs. 0. 605 ±0. 367) , p-Smad2/3 ( 1. 457 ±0. 111 vs. 0. 815 ±0. 039) , and p-Smad3 ( 1. 320 ±0. 147 vs. 0. 623 ±0. 128) increased with statistical significance ( P lt; 0. 01) . Meanwhile the expression of Smad7 reduced ( 0. 614 ±0. 107 vs. 0. 865 ±0. 063, P lt;0. 05) . But in the TGF-β1 + BP5 treatment groups, over-expressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by BP5, especially at the BP5 concentration of 10 μg/mL ( collagen-Ⅰ: 0. 718 ±0. 049 vs. 1. 402 ±0. 158; p-Smad2 /3: 0. 696 ±0. 031 vs. 1. 457 ±0. 111; p-Smad3: 0. 766 ±0. 006 vs. 1. 320 ±0. 147; all P lt; 0. 01) . Otherwise, the up-regulation of Smad7 ( 1. 237 ±0. 173 vs. 0. 614 ±0. 107) was found.Conclusions Bursopentin can reduce the expressions of collagen-Ⅰ and α-SMA protein of fibroblast stimulated by TGF-β1 , maybe through inhibiting TGF-β1 /Smads transduction pathway. It is suggested that bursopentin may have intervention effect on pulmonary fibrosis.
ObjectiveTo investigate the effects of liver X receptor agonist, T0901317, on the proliferation, migration and hydroxyproline production of human embryonic lung fibroblasts (HELF). MethodsHELF cells were devided into a control group, a growth factor group, a T0901317 group and three growth factor+T0901317 groups. The cells of the control group were treated with Dulbecco's modified Eagle medium. The cells of T0901317 group were treated with 1.00 μmol/L T0901317. The growth factor+T0901317 groups were incubated with different doses of T0901317 (0.25 μmol/L, 0.50 μmol/L and 1.00 μmol/L) for 2 h. Then the cells of the growth factor+T0901317 groups and the growth factor group were incubated with basic fibroblast growth factor and transforming growth factor-β1 for 24 h. The proliferation, migration and collagen production of HELF were determined by cell counting kit-8 method, transwell chamber, and hydroxyproline method. ResultsCompared with the control group, T0901317 had no effect on the proliferation, migration and hydroxyproline production of HELF. Growth factors could promote the proliferation, migration and hydroxyproline production of HELF significantly. T0901317 could inhibit those effects of growth factors with a dosage-dependent manner. ConclusionT0901317 may inhibit the proliferation, migration and hydroxyproline production of HELF induced by growth factors.
目的 探讨经转化生长因子-β1 ( TGF-β1) 基因修饰的未成熟树突状细胞(imDC) 预处理大鼠小肠移植受体后的外周血及移植肠浸润T 细胞的变化及意义。方法 选用近交系F344/ N 和BN 大鼠建立全小肠异位移植模型,实验分4 组(每组24 只) : 同基因移植组(BN-BN 组) 、异基因移植组( F344/ N-BN 组) 、异基因移植+ TGF-β1 基因转染imDC 组( F344/ N-BN + TGF2β1 组) 和异基因移植+ TGF-β1 基因转染imDC + FK506 组( F344/ N-BN + TGF-β1 + FK506 组) 。各组大鼠分别于术后3 、5 、7 d 各处死6 只,获取大鼠静脉血和移植肠。应用免疫组化SABC 法检测受体鼠外周血及移植肠CD4 + 、CD8 + 、CD25 + 细胞和IL-4 的表达。同时行移植肠组织病理学检查并观察大鼠生存情况。结果 TGF-β1 修饰的DC 细胞能显著抑制外周血及移植肠浸润淋巴细胞CD4 + 、CD8 + 及CD25 + 的表达,并提高IL-4 的表达; 显著延长受体大鼠的生存时间,但移植肠仍有排斥反应的病理组织学征象。结论 TGF-β1 修饰的DC 通过影响受体外周血及移植肠浸润T 细胞对大鼠小肠移植发挥免疫抑制作用。
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 investigate the effects of caveolin-1 scaffolding domain peptide ( CSD-p)on expressions of extracellular matrix and Smads in human fetal lung fibroblasts. Methods Human fetal lung fibroblasts were cultured in vitro and divided into four groups. A control group: the cells were cultured in DMEMwithout TGF-β1 or CSD-p. A CSD-p treatment group: the cells were cultured in DMEMcontaining 5 μmol /L CSD-p. A TGF-β1 treatment group: the cells were cultured in DMEMcontaining 5 μg/L TGF-β1 .A TGF-β1 + CSD-p treatment group: the cells were cultured in DMEM containing 5 μg/L TGF-β1 and 5 μmol /L CSD-p. Caveolin -1 mRNA was detected by RT-PCR. Caveolin-1, collagen-Ⅰ, α-SMA, p-Smad2,p-Smad3 and Smad7 proteins were measured by Western blot. Results Compared with the control group,the Caveolin -1 mRNA and protein expressions in the cells of TGF-β1 group significantly reduced ( mRNA:0. 404 ±0. 027 vs. 1. 540 ±0. 262; protein: 0. 278 ±0. 054 vs. 1. 279 ±0. 085; P lt; 0. 01) , and the expression levels of collagen-Ⅰ and α-SMA proteins significantly increased ( collagen-Ⅰ: 1. 127 ±0. 078 vs.0. 234 ±0. 048; α-SMA: 1. 028 ±0. 058 vs. 0. 295 ±0. 024) . Meanwhile, the expression levels of p-Smad2 ( 1. 162 ±0. 049 vs. 0. 277 ±0. 014) and p-Smad3 proteins ( 1. 135 ±0. 057 vs. 0. 261 ±0. 046) increased with statistical significance ( P lt; 0. 01) , but the expression level of Smad7 protein significantly reduced( 0. 379 ±0. 004 vs. 1. 249 ±0. 046, P lt;0. 001) . In the CSD-p group, CSD-p had no significant effects on the expressions of above proteins compared with the control group. But in the TGF-β1 +CSD-p group, the overexpressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by CSD-p ( collagen-Ⅰ: 0. 384 ±0. 040 vs. 1. 127 ±0. 078; α-SMA: 0. 471 ±0. 071 vs. 1. 127 ±0. 078;p-Smad2: 0. 618 ±0. 096 vs. 1. 162 ±0. 049; p-Smad3: 0. 461 ±0. 057 vs. 1. 135 ±0. 057; P lt; 0. 01) .Otherwise, the up-regulation of Smad7 ( 0.924 ±0. 065 vs. 0.379 ±0. 004) was found. Conclusions CSD-p can reduce fibroblast collagen-I and α-SMA protein expressions stimulated by TGF-β1 , possibly through regulation of TGF-β1 /Smads signaling pathway. It is suggested that an increase in caveolin -1 function through the use of CSD-p may be an intervention role in pulmonary fibrosis.
Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
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.