Objective To investigate the regulatory effect of multifactor on the matrix metalloproteinases-9 （MMP-9） and the tissue inhibitor of metalloproteinase-1 （TIMP-1） in endometrial stromal cells. Methods The endometri...Objective To investigate the regulatory effect of multifactor on the matrix metalloproteinases-9 （MMP-9） and the tissue inhibitor of metalloproteinase-1 （TIMP-1） in endometrial stromal cells. Methods The endometrial stromal cells separated from the proliferative endometrial tissues were incubated with medium alone, 17-β estradiol （E2,10^-8 mol/L）, medroxyprogesterone acetate （MPA, 10^-6 mol/L）, E2（10^-8 mol/L）＋MPA （10^-6 mol/L）, E2 （10^-8 mol/L）＋MPA （10^-6 mol/L）＋RU486 （10^-5 mol/L） or HB-EGF （10 ng/ml） for 48 h respectively. The expressions of MMP-9 and TIMP-1 were detected by in situ hybridization, immunocytochemistry, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. Results Compared with control group [mRNA, 0. 729 ± 0. 090 （MMP-9） and 1.056± 0.154 （TIMP-1）; protein, 0.545 ±0.086 （MMP-9） and 0.745 ±0.154 （TIMP-1）], expressions of MMP-9 and TIMP-1 in E2 alone, progestin alone or E2 combined with progestin group were respectively：mRNA, 0.413 ± 0.069, 0.402 ± 0.073 and 0.407 ± 0.039; 0.487 ± 0.093, 0.503 ± 0.093 and 0.468 ± 0.075：protein, 0.294 ± 0.076, 0.331 ±0.064 and 0.265 ±0.049; 0.425 ±0.085, 0.397 ±0.065 and 0.435 ± 0.099. RU486 weakened the expression level of down-regulation, while HB-EGF elevated the level of MMP-9 and TIMP-1 after 48 h treatment （mRNA, 0.955 ± 0.068 and 1.396 ± 0.238; protein, 0. 780 ± 0.109 and 0.985 ± 0.165）. Conclusions 1） Both E2 and progestin can down-regulate the expressions of MMP-9 and TIMP-1 in endometrial stromal cells, but RU486 can inhibit the effect. 2） HB-EGF can elevate the level of MMP-9 and TIMP-1. 3） E2, progestin and HB-EGF have effect on the ratio of MM-P/TIMP-1.展开更多
目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂...目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂量组及骨瓜大剂量组,分别于模型组、骨瓜小剂量组、骨瓜大剂量组大鼠右膝关节腔内多点注射小牛软骨Ⅱ型胶原加福氏完全佐剂0.1 m L,制备类风湿性关节炎动物模型,造模成功后,骨瓜小剂量组、骨瓜大剂量组分别用骨瓜提取物注射液肌注治疗21 d,于治疗第1、7、14、21天,分别测定四组大鼠关节积液中HA及IL-1β含量;检测滑膜BMPs及MMP-1阳性表达,测量右足趾关节以下部位的容积并计算足肿胀度,记录各组动物消肿时间,评价骨瓜提取物注射液对大鼠类风湿性关节炎的治疗效果。结果治疗下14、21 d时,骨瓜小剂量组、骨瓜大剂量组MMP-1阳性表达及关节积液中IL-1β明显降低,BMPs及HA含量明显升高,消肿时间缩短,足肿胀度明显优于模型组,与模型组比较,差异有统计学意义(P<0.05),骨瓜小剂量组、骨瓜大剂量组间各指标差异无统计学意义(P>0.05)。结论骨瓜提取物注射液可促进类风湿关节炎模型大鼠关节积液HA及BMPs分泌与合成,降低软骨细胞MMP-1阳性表达,抑制炎性介质IL-1β对关节滑膜的侵蚀而起到保护关节的作用。展开更多
The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basi...The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.展开更多
The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor(TGF)-β_(1)genes in bo...The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor(TGF)-β_(1)genes in bone marrow-derived mesenchymal stem cells(MSCs)in vitro.The full-length rat TGF-β_(1)cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog.The transient and stable expression of TGF-β_(1)by MSCs was detected by using immunohistochemical staining.The lipofectamine-mediated gene therapy efficiently transfected MSCs in vitro with the TGF-β_(1)gene causing a marked up-regulation in TGF-β_(1)expression as compared with the vector-transfected control groups,and the increased expression persisted for at least 4 weeks after selected with G418.It was suggested that bone marrow-derived MSCs were susceptible to in vitro lipofectamine mediated TGF-β_(1)gene transfer and that transgene expression persisted for at least 4 weeks.Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology,an innovative concept,i.e.molecular tissue engineering,are put forward for the first time.As a new branch of tissue engineering,it represents both a new area and an important trend in research.Using this technique,we have a new powerful tool with which:(1)to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and(2)to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis.展开更多
Objective To study the effect and possible mechanism of Ligustrazine-a chinese traditional herbal medicine （CTM） on rat osteoarthritis （OA）. Methods OA model was surgically induced. Morphology of articular cartila...Objective To study the effect and possible mechanism of Ligustrazine-a chinese traditional herbal medicine （CTM） on rat osteoarthritis （OA）. Methods OA model was surgically induced. Morphology of articular cartilage was done by HE staining and mankin score was calculated; interleukin-1 and TNFα activity of rat joints was determined by 3-（4,5 dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） method; immunohistochemistry of MMP-13 and Cathepsin K was done by ABC method while the mRNA level for MMP-13, cathepsin K and tissue inhibitor of matrix metalloproteinase 1 （TIMP-1） was evaluated by RT-PCR method. ResultsLigustrazine （60 mg·kg^-1·d^-1 or 30 mg·kg^-1·d^-1 , but not 10 mg·kg^-1·d^-1 , im, ×8 weeks） reduced morphological changes of articular cartilage of OA rats. IL-1 and TNFα production of OA joints increases, together with MMP-13, cathepsine K and their mRNA levels in articular cartilage. The administration of Ligustrazine （60 mg·kg^-1·d^-1 -1 or 30 mg·kg^-1·d^-1 , im, ×8 weeks） reduced above changes significantly. At the same time, tissue inhibitor of metrix metalloprotease （TIMP1） mRNA level increased also in OA rats while Ligustrazine （60 mg·kg^-1·d^-1 , 30 mg·kg^-1·d^-1 or 10 mg·kg^-1·d^-1 , im, ×8 weeks） had no significant effect on it. Conclusion Ligustrazine （60 mg·kg^-1·d^-1 or 30 mg·kg^-1·d^-1 , but not 10 mg·kg^-1·d^-1 , im, ×8 weeks） effectively delays articular cartilage degradation of OA rats and could be a potential drug candidate for OA treatment.展开更多
文摘Objective To investigate the regulatory effect of multifactor on the matrix metalloproteinases-9 （MMP-9） and the tissue inhibitor of metalloproteinase-1 （TIMP-1） in endometrial stromal cells. Methods The endometrial stromal cells separated from the proliferative endometrial tissues were incubated with medium alone, 17-β estradiol （E2,10^-8 mol/L）, medroxyprogesterone acetate （MPA, 10^-6 mol/L）, E2（10^-8 mol/L）＋MPA （10^-6 mol/L）, E2 （10^-8 mol/L）＋MPA （10^-6 mol/L）＋RU486 （10^-5 mol/L） or HB-EGF （10 ng/ml） for 48 h respectively. The expressions of MMP-9 and TIMP-1 were detected by in situ hybridization, immunocytochemistry, reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. Results Compared with control group [mRNA, 0. 729 ± 0. 090 （MMP-9） and 1.056± 0.154 （TIMP-1）; protein, 0.545 ±0.086 （MMP-9） and 0.745 ±0.154 （TIMP-1）], expressions of MMP-9 and TIMP-1 in E2 alone, progestin alone or E2 combined with progestin group were respectively：mRNA, 0.413 ± 0.069, 0.402 ± 0.073 and 0.407 ± 0.039; 0.487 ± 0.093, 0.503 ± 0.093 and 0.468 ± 0.075：protein, 0.294 ± 0.076, 0.331 ±0.064 and 0.265 ±0.049; 0.425 ±0.085, 0.397 ±0.065 and 0.435 ± 0.099. RU486 weakened the expression level of down-regulation, while HB-EGF elevated the level of MMP-9 and TIMP-1 after 48 h treatment （mRNA, 0.955 ± 0.068 and 1.396 ± 0.238; protein, 0. 780 ± 0.109 and 0.985 ± 0.165）. Conclusions 1） Both E2 and progestin can down-regulate the expressions of MMP-9 and TIMP-1 in endometrial stromal cells, but RU486 can inhibit the effect. 2） HB-EGF can elevate the level of MMP-9 and TIMP-1. 3） E2, progestin and HB-EGF have effect on the ratio of MM-P/TIMP-1.
文摘目的观察骨瓜提取物注射液对大鼠类风湿关节炎关节滑膜骨形态发生蛋白(BMPs)、软骨细胞MMP-1阳性表达及关节积液透明质酸(HA)、白细胞介素-1β亚型(IL-1β)的影响,探讨其作用机制。方法大鼠40只,随机分为空白对照组、模型组、骨瓜小剂量组及骨瓜大剂量组,分别于模型组、骨瓜小剂量组、骨瓜大剂量组大鼠右膝关节腔内多点注射小牛软骨Ⅱ型胶原加福氏完全佐剂0.1 m L,制备类风湿性关节炎动物模型,造模成功后,骨瓜小剂量组、骨瓜大剂量组分别用骨瓜提取物注射液肌注治疗21 d,于治疗第1、7、14、21天,分别测定四组大鼠关节积液中HA及IL-1β含量;检测滑膜BMPs及MMP-1阳性表达,测量右足趾关节以下部位的容积并计算足肿胀度,记录各组动物消肿时间,评价骨瓜提取物注射液对大鼠类风湿性关节炎的治疗效果。结果治疗下14、21 d时,骨瓜小剂量组、骨瓜大剂量组MMP-1阳性表达及关节积液中IL-1β明显降低,BMPs及HA含量明显升高,消肿时间缩短,足肿胀度明显优于模型组,与模型组比较,差异有统计学意义(P<0.05),骨瓜小剂量组、骨瓜大剂量组间各指标差异无统计学意义(P>0.05)。结论骨瓜提取物注射液可促进类风湿关节炎模型大鼠关节积液HA及BMPs分泌与合成,降低软骨细胞MMP-1阳性表达,抑制炎性介质IL-1β对关节滑膜的侵蚀而起到保护关节的作用。
基金This project was supported by a grant from NationalNatural Science Foundation of China (No. 30 170 2 70 )
文摘The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.
文摘The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor(TGF)-β_(1)genes in bone marrow-derived mesenchymal stem cells(MSCs)in vitro.The full-length rat TGF-β_(1)cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog.The transient and stable expression of TGF-β_(1)by MSCs was detected by using immunohistochemical staining.The lipofectamine-mediated gene therapy efficiently transfected MSCs in vitro with the TGF-β_(1)gene causing a marked up-regulation in TGF-β_(1)expression as compared with the vector-transfected control groups,and the increased expression persisted for at least 4 weeks after selected with G418.It was suggested that bone marrow-derived MSCs were susceptible to in vitro lipofectamine mediated TGF-β_(1)gene transfer and that transgene expression persisted for at least 4 weeks.Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology,an innovative concept,i.e.molecular tissue engineering,are put forward for the first time.As a new branch of tissue engineering,it represents both a new area and an important trend in research.Using this technique,we have a new powerful tool with which:(1)to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and(2)to affect a better repair of full-thickness articular cartilage defects that occur as a result of injury and osteoarthritis.
文摘Objective To study the effect and possible mechanism of Ligustrazine-a chinese traditional herbal medicine （CTM） on rat osteoarthritis （OA）. Methods OA model was surgically induced. Morphology of articular cartilage was done by HE staining and mankin score was calculated; interleukin-1 and TNFα activity of rat joints was determined by 3-（4,5 dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） method; immunohistochemistry of MMP-13 and Cathepsin K was done by ABC method while the mRNA level for MMP-13, cathepsin K and tissue inhibitor of matrix metalloproteinase 1 （TIMP-1） was evaluated by RT-PCR method. ResultsLigustrazine （60 mg·kg^-1·d^-1 or 30 mg·kg^-1·d^-1 , but not 10 mg·kg^-1·d^-1 , im, ×8 weeks） reduced morphological changes of articular cartilage of OA rats. IL-1 and TNFα production of OA joints increases, together with MMP-13, cathepsine K and their mRNA levels in articular cartilage. The administration of Ligustrazine （60 mg·kg^-1·d^-1 -1 or 30 mg·kg^-1·d^-1 , im, ×8 weeks） reduced above changes significantly. At the same time, tissue inhibitor of metrix metalloprotease （TIMP1） mRNA level increased also in OA rats while Ligustrazine （60 mg·kg^-1·d^-1 , 30 mg·kg^-1·d^-1 or 10 mg·kg^-1·d^-1 , im, ×8 weeks） had no significant effect on it. Conclusion Ligustrazine （60 mg·kg^-1·d^-1 or 30 mg·kg^-1·d^-1 , but not 10 mg·kg^-1·d^-1 , im, ×8 weeks） effectively delays articular cartilage degradation of OA rats and could be a potential drug candidate for OA treatment.
文摘目的探讨应用白介素-1受体拮抗蛋白(IL1RA)对早期创伤性关节炎软骨细胞代谢的影响,主要观察应用后相关细胞炎性因子及蛋白酶基因的表达变化。方法 44只健康成年新西兰大白兔,随机分为对照(假手术)组(CON组,n=4)、安慰剂组(PBS组,n=20)、IL1RA治疗组(IL1RA组,n=20)。其中PBS组及IL1RA组均接受关节内重物击打,造成创伤性关节炎模型。造模后1 h,CON组不接受任何治疗,PBS组及IL1RA组实验兔膝关节则分别给予0.5 m L无菌PBS和0.5 m L IL1RA-PBS关节腔注射。术后3 h及8 h采集关节液,检测IL-1β含量。术后8 h处死动物取软骨组织进行软骨细胞培养后,测定细胞活力及凋亡率,RT-PCR检测软骨组织中ADAMTs-4、ADAMTs-5、MMP-3、IL-1β及TNF-α的m RNA表达。结果术后3 h及8 h,PBS组关节液中IL-1β的含量均明显高于CON组(P<0.01)。然而,经IL1RA介入治疗后,IL-1β在术后各测试点的含量均明显低于PBS组(P<0.05)。与CON组相比,术后8h培养的PBS组软骨细胞活力明显降低(P<0.05),而IL1RA组软骨细胞活力与CON组相比无显著性差异,但明显高于PBS组。另一方面,与CON组及IL1RA组相比,PBS组的细胞凋亡率明显增高(P<0.05)。而IL1RA组与CON组相比,细胞凋亡率无明显差别(P>0.05)。RT-RCR结果显示,与CON组比较,PBS组所有目的基因m RNA的表达均明显增加(P<0.01)。然而,IL1RA组所有目的基因的表达与CON组相比无统计学差异,但明显低于PBS组(P<0.01)。结论实验结果表明创伤后关节炎早期应用IL1RA介入治疗可有效降低关节液细胞炎性因子的浓度,抑制细胞炎性因子对软骨细胞生长增殖的负性影响,降低细胞凋亡,且对于促进软骨细胞基质降解的细胞炎性因子及蛋白酶的表达显著下调,对PTOA病程发展具有抑制作用。
