integrin β_1在不同细胞周期肝癌细胞与内皮细胞粘附中的作用

探讨了integrinβ1在不同细胞周期人肝癌细胞(SMMC-7721)上的表达和在肝癌细胞与人脐静脉内皮细胞粘附过程中的作用。未同步处理的肝癌细胞(对照组)各细胞周期时相百分比为G0/G1期53.51%、G2/M期11.01%、S期35.48%,采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法获得G1期和S期的肝癌细胞,其同步率分别为74.09%和98.29%。G1期肝癌细胞integrinβ1表达的荧光强度较S期和对照组相应值明显降低。利用微管吸吮技术定量研究了肝癌细胞与内皮细胞之间的粘附力学特性,发现G1期肝癌细胞的粘附力值比S期相应值明显降低(P<0.01),而S期的粘附力值与对照组比较无明显差别,integrinβ1在肝癌细胞与内皮细胞粘附过程中的贡献约50%。结果提示:胸腺嘧啶脱氧核苷和秋水仙碱能较好地将肝癌细胞同步于G1期和S期,integrinβ1在SMMC-7721肝癌细胞上的表达水平呈现周期差异,在肝癌细胞与内皮细胞粘附过程中,S期细胞可能起的作用更大,integrinβ1在这一粘附过程中起着重要作用。

Correlation of integrin β3 mRNA and vascular endothelial growth factor protein expression profiles with the clinicopathological features and prognosis of gastric carcinoma

AIM： To investigate integrin β3 mRNA and vascular endothelial growth factor （VEGF） protein expression in gastric carcinoma, and its correlation with microvascular density, growth-pattern, invasion, metastasis and prognosis. METHODS： In situ hybridization（ISH） of integrin β3 mRNA and immunohistochemistry of VEGF and CD34 protein were performed on samples from 118 patients with gastric cancer. RESULTS： The positive rate of integrin β3 mRNA in non-tumor gastric mucosa （20%） was significantly lower than that of the gastric cancer tissue （52.5%, X^2 = 10.20, P 〈 0.01）. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of integrin β3 mRNA were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of VEGF protein were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the mean MVD were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. It was found that the positive expression rate of integrin β3 mRNA was positively related to that of VEGF protein （P 〈 0.01） and MVD （P 〈 0.05）, meanwhile the positive expression rate of VEGF protein was positively related to MVD （P 〈 0.05）. The mean survival period in patients with positive expression of integrin β3 mRNA and VEGF, and MVD ≥54.9/mm^2 was significantly shorter than that in patients with negative expression of integrin β3 mRNA （P 〈 0.05） and VEGF （P 〈 0.01）, and MVD 〈 54.9/mm^2 （P 〈 0.01）. Five-year survival rate in patients with positive expression of integrin β3 mRNA and VEGF, and MVD ≥54.9/mm^2 was significantly lower than those with negative expression of integrin β3 mRNA （P 〈 0.05）, VEGF （P 〈 0.05）, and MVD 〈 54.9/mm^2 （P 〈 0.01）. CONCLUSION： Integrin β3 and VEGF expression can synergistically enhance tumor angiogenesis, and may play a crucial role in invasion and metastasis of gastric carcinoma. Therefore, they may be prognostic biomarkers and novel molecular therapeutic targets.

癌相关成纤维细胞中Gal-1表达通过integrin β1促进胃癌细胞侵袭的机制研究

目的研究癌相关成纤维细胞中Gal-1表达通过integrinβ1促进胃癌细胞侵袭的机制。方法高表达Gal-1的胃癌CAFs采用原代培养法,通过CAFs与胃癌细胞共培养、siRNA转染、抑制Gal-1/封闭integrinβ1表达、细胞侵袭能力实验、侵袭能力影响实验和IHC实验等技术,研究胃癌细胞侵袭迁移能力受胃癌CAFs中Gal-1表达的影响程度及作用机制。分析高表达Gal-1的CAFs对癌细胞迁移能力和侵袭能力的影响,比较Gal-1和integrinβ1免疫组化实验结果。结果 CAFs共培养使得胃癌细胞侵袭和迁移能力得到明显提升,Gal-1的表达遭到siRNA抑制后,CAFs对迁移能力和侵袭能力的促进作用得到有效抑制。Gal-1主要在胃癌细胞的间质成纤维细胞中表达,integrinβ1主要在胃癌细胞的细胞膜中表达。Gal-1和integrinβ1在胃癌中的阳性率分别为56.67%(51/90)、43.33%(39/90),并且其表达与淋巴结转移、远处转移、TNM分期差异具有统计学意义(P<0.05)。结论CAFs表达Gal-1并通过促进胃癌细胞integrinβ1表达的形式提高胃癌细胞的侵袭迁移能力,指明今后胃癌的治疗和相关预后的研究方向,表现出一定的理论研究价值。

17β-雌二醇对机械牵拉诱导心肌细胞integrin β1/FAK/p38 MAPK信号转导的影响

目的:研究17β-雌二醇(17β-estradiol,E2)对体外机械牵拉诱导心肌细胞integrinβ1/FAK/p38 MAPK信号转导的影响。方法:以机械牵拉刺激体外培养的新生大鼠心肌细胞,建立心肌细胞肥大模型,采用免疫共沉淀方法检测integrinβ1和FAK的结合情况,Western blot方法检测FAK和p38 MAPK磷酸化水平的变化。结果:机械牵拉心肌细胞24 h后,integrinβ1和FAK的结合显著增加,FAK和p38 MAPK磷酸化水平亦明显增强。100 nmol/L E2预处理30 min可明显减轻机械牵拉诱导的心肌细胞integrinβ1和FAK的结合增加,抑制FAK和p38 MAPK磷酸化的水平增强,该效应可被雌激素受体非特异性拮抗剂ICI182780逆转。结论:100 nmol/L的E2能够抑制机械牵拉诱导心肌细胞肥大发生发展过程中integrinβ1对其下游FAK招募结合增加,降低FAK及p38MAPK的磷酸化活性,提示E2与雌激素受体结合后通过抑制integrinβ1/FAK/p38 MAPK信号转导途径的激活,从而发挥心血管保护作用。

Adhesion of different cell cycle human hepatoma cells to endothelial cells and roles of integrin β_1

AIM: To investigate the adhesive mechanical properties of different cell cycle human hepatoma cells (SMMC-7721) to human umbilical vein endothelial cells (ECV-304), expression of adhesive molecule integrinβ1 in SMMC-7721 cells and its contribution to this adhesive course. METHODS: Adhesive force of SMMC-7721 cells to endothelial cells was measured using micropipette aspiration technique. Synchronous G1 and S phase SMMC-7721 cells were achieved by thymine-2-deoxyriboside and colchicines sequential blockage method and double thymine-2-deoxyriboside blockage method, respectively. Synchronous rates of SMMC-7721 cells and expression of integrinβ1 in SMMC-7721 cells were detected by flow cytometer. RESULTS: The percentage of cell cycle phases of general SMMC-7721 cells was 11.01% in G2/M phases, 53.51% in G0/G1 phase, and 35.48% in S phase. The synchronous rates of G1 and S phase SMMC-7721 cells amounted to 74.09% and 98.29%, respectively. The adhesive force of SMMC-7721 cells to endothelial cells changed with the variations of adhesive time and presented behavior characteristics of adhesion and de-adhesion. S phase SMMC-7721 cells had higher adhesive forces than d phase cells [(307.65±92.10)×10-10N vs(195.42±60.72)×10-10N, P<0.01]. The expressive fluorescent intensity of integrinβ1 in G1 phase SMMC-7721 cells was depressed more significantly than the values of S phase and general SMMC-7721cells. The contribution of adhesive integrinβ1 was about 53% in this adhesive course. CONCLUSION: SMMC-7721 cells can be synchronized preferably in d and S phases with thymine-2-deoxyriboside and colchicines. The adhesive molecule integrinβ1 expresses a high level in SMMC-7721 cells and shows differences in various cell cycles, suggesting integrin β1 plays an important role in adhesion to endothelial cells. The change of adhesive forces in different cell cycle SMMC-7721 cells indicates that S phase cells play predominant roles possibly while they interact with endothelial cells.

妊娠期高血压疾病患者胎盘组织中基质金属蛋白酶-9(MMP-9)和整合素β 3(Integrin β 3)的表达

目的研究基质金属蛋白酶-9(MMP-9)和整合素β3(Integrin β3)在妊娠期高血压疾病患者胎盘组织中的表达,并探讨其与妊娠期高血压疾病发病的关系及两者的相关性。方法采用免疫组织化学染色(SP法)分别检测In-tegrinβ3、MMP-9在10例妊娠期高血压疾病患者及10例正常妊娠者胎盘组织中的表达。结果妊娠期高血压疾病组胎盘Integrinβ3表达明显高于正常妊娠组(P<0.01)。妊娠期高血压疾病组胎盘MMP-9表达明显低于正常妊娠组(P<0.01),且随病情的加重MMP-9的表达有下降趋势,有统计学意义(P<0.05)。结论本研究显示:与正常妊娠组比较妊娠期高血压疾病患者胎盘组织integrinβ3表达明显增加、MMP-9的表达明显减少,且随病情加重有减少趋势。表明integrinβ3、MMP-9可能参与妊娠期高血压疾病的发生和发展。

Inhibitory Effects of Alpha-zearalenol on Angiotensin II-Induced Integrin β_3 mRNA via Suppression of Nuclear Factor-κB

To investigate the effect of α-zearalenol on angiotensin If-induced β3 integrin mRNA expression in human umbilical vein endothelial cells （HUVECs）. Methods The mRNA level in integrin β3 was determined by reverse transcription-polymerase chain reaction. Endothelial NF-κB activity was determined by the luciferase activity assay of plasmid NF-κB-LUC. Results The angiotensin Ⅱ- induced β3 integrin mRNA expression was inhibited by α-zearalenol and 17β-estradiol （10nmol/L ^-1 μmol/L）, but not influenced by ICI 182, 780, a pure competitive antagonist for estrogen receptor or a nitric oxide inhibitor N^ω-Nitro-L-arginine methyl ester hydrochloride. Alpha-zearalenol and 17β-estradiol suppressed the angiotensin Ⅱ-induced activation of NF-κB in endothelial cells. Condusion Alpha-zearalenol inhibits angiotensin Ⅱ-induced integrin β3 mRNA expression by suppressing NF-κB activation in endothelial cells.

Integrin β1、Cyclin D1在胃癌中的表达及其临床意义

目的:研究细胞周期素D1(cyclin D1)及整合素β1(integrin β1)在胃癌组织中的表达及其与胃癌生物学行为的关系。方法:用免疫组化SP法,检测60例胃癌组织中整合素β1、细胞周期素D1蛋白的表达水平。结果:整合素β1的表达与胃癌的浸润深度及有、无淋巴结转移有关,浸润程度越深阳性表达率越高;有淋巴结转移组明显高于无淋巴结转移组(P<0.01),其在胃癌中的表达与浸润程度、TNM分期及淋巴结转移相关;与病理学分级、患者年龄、性别无关。细胞周期素D1表达与胃癌分化程度、分期、浸润深度及有、无淋巴结转移有关。细胞周期素D1和整合素β1在胃癌组织中表达呈正相关。结论:整合素β1的表达与胃癌的侵袭、转移有关,在一定程度上可反映胃癌病人的预后。细胞周期素D1蛋白表达与胃癌病理组织学分级相关,其过表达可能与胃癌的发生有关。检测细胞周期素D1和整合素β1的表达可以为判断胃癌有无淋巴结转移起一定的提示作用。

Integrin β1对胃癌细胞SGC7901多药耐药性的影响

目的:探究整合素β1(integrinβ1)对胃癌多药耐药性的影响及可能的作用机制。方法:Western blot法及q PCR实验检测胃癌细胞株SGC-7901及胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达情况。采用integrinβ1反义寡核苷酸转染,敲减胃癌耐药细胞株SGC-7901/DDP中integrinβ1的表达,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Western blot法检测integrinβ1、Bcl-2/Bax、cleaved caspase-3/caspase-3、细胞色素C(CytC)和p-AKT/AKT的蛋白水平。结果:耐药细胞株SGC7901/DDP中integrinβ1的mRNA及蛋白表达水平均明显高于亲本细胞株;并且在亲本细胞株SGC7901中加入顺铂、长春新碱及5-氟尿嘧啶等化疗药物刺激后,integrinβ1的蛋白表达水平明显升高。敲减integrinβ1的表达可诱导胃癌耐药细胞SGC7901/DDP的凋亡,增加细胞对化疗药物的敏感性;此外下调Bcl-2/Bax、p-AKT^(Ser473)和p-AKT^(Thr308)的蛋白水平,同时促进线粒体Cyt-C的释放,上调cleaved caspase-3的蛋白水平。结论:敲减胃癌顺铂耐药细胞SGC7901/DDP的integrinβ1表达可恢复细胞对化疗药物的敏感性,促进细胞经线粒体路径的凋亡,其机制可能与抑制AKT的磷酸化,阻断该信号通路有关。

integrin β_1在胰腺癌中的表达及临床意义

目的:观察integrinβ1在胰腺癌及癌旁组织中表达,与临床病理特征参数进行相关性分析,初步探讨其临床意义。方法:应用免疫组化技术(SABC法)检测integrinβ1在25例胰腺癌及其癌旁组织中的表达,图像分析软件分析其表达强度,分析与胰腺癌临床病理特征参数之间的关系。结果:integrinβ1在胰腺癌及其癌旁组织中的高表达率分别为76.00%、40.00%,两者差异有统计学意义(P<0.05)。在胰腺癌组织中,integrinβ1的表达强度和临床分期及有无淋巴结转移有关(P<0.05,P<0.05),与性别、年龄、肿瘤部位、大小及分化程度无关。结论:integrinβ1可以作为胰腺癌新的分子病理标志物,可能为胰腺癌的基因治疗提供新的靶点。

Role of integrin β1 in sensitivity to chemotherapy of pulmonary adenocarcinoma A549

Objective:The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.Methods:Human pulmonary adenocarcinoma A549 multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Cell counting using blood cell counter was employed to detect the sensitivity to ADM of A549 MCS before and after blocking integrin β1;integrin β1 expression of A549 MCS and cell apoptosis was detected by flow cytometry.Results:A549 MCS were successfully established.The integrin β1 expression of A549 MCS elevated with the concentration of ADM(< 0.02 μg/mL).Blocking of integrin β1 lead to higher sensitivity to ADM,and IC50 decreased from 0.19 μg/mL to 0.11 μg/mL,and apoptosis rate increased from(15.81 ± 1.87)% to(30.14 ± 2.89)%.Conclusion:The cell adhesion molecules integrin β1 could influence the sensitivity to chemotherapy of A549 MCS and inhibiting of cell apoptosis might be its mechanism.

Collagen type Ⅱ suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin β1-SMAD1 interaction

Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage.Collagen type II(COL2A1)was previously considered to be only a structural component of the cartilage matrix,but recently,it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy.However,the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear.In our study,a Col2a1 p.Gly1170Ser mutant mouse model was constructed,and Col2a1 loss was demonstrated in homozygotes.Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein(BMP)-SMAD1 pathway.Upon interacting with COL2A1,integrinβ1(ITGB1),the major receptor for COL2A1,competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import.COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and,through ERK1/2-SMAD1 interaction,it further repressed SMAD1 activation,thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy.Moreover,COL2A1 expression was downregulated,while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage.Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.

Silencing profilin-1 inhibits gastric cancer progression via integrin β1/focal adhesion kinase pathway modulation

AIM:To investigate the role of profilin-1(PFN1)in gastric cancer and the underlying mechanisms.METHODS:Immunohistochemical analysis,quan-titative real-time polymerase chain reaction(q RTPCR)and Western blot were performed to detect PFN1expression in clinical gastric carcinoma and adjacent tissues,and the association of PFN1 expression with patient clinicopathological characteristics was analyzed.PFN1 was knocked down to investigate the role of this protein in cell proliferation and metastasis in the SGC-7901 cell line.To explore the underlying mechanisms,the expression of integrinβ1 and the activity of focal adhesion kinase(FAK)and the downstream proteins extracellular-regulated kinase(ERK)1/2,P38 mitogen-activated protein kinase(MAPK),phosphatidylinositol 3-kinase(PI3K),AKT and mammalian target of rapamycin(m TOR)were measured through Western blot or q RT-PCR analysis.Fibronectin(FN),a ligand of integrinβ1,was used to verify the correlation between alterations in the integrinβ1/FAK pathway and changes in tumor cell aggressiveness upon PFN1 perturbation.RESULTS:Immunohistochemical,Western blot and q RT-PCR analyses revealed that PFN1 expression was higher at both the protein and m RNA levels in gastric carcinoma tissues compared with the adjacent tissues.In addition,high PFN1 expression(53/75,70.4%)was correlated with tumor infiltration,lymph node metastasis and TNM stage in gastric cancer,but not with gender,age,location,tumor size,or histological differentiation.In vitro experiments showed that PFN1knockdown inhibited the proliferation of SGC-7901cells through the induction G0/G1 arrest.Silencing PFN1 inhibited cell migration and invasion and downregulated the expression of matrix metalloproteinase(MMP)-2 and MMP9.Moreover,silencing PFN1 reduced the expression of integrinβ1 at the protein level and inhibited the activity of FAK,and the downstream effectors ERK1/2,P38MAPK,PI3K,AKT and m TOR.FN-promoted cell proliferation and metastasis via the integrinβ1/FAK pathway was ameliorated by PFN1silencing.CONCLUSION:These findings suggest that PFN1 plays a critical role in gastric carcinoma progression,and these effects are likely mediated through the integrinβ1/FAK pathway.

MMP-2与integrin β_1在脑胶质瘤中的表达及其意义

目的 探讨MMP 2与integrinβ1在胶质瘤中的表达及相互关系。方法 采用S P法对胶质瘤组织进行MMP 2和integrinβ1单克隆抗体的免疫组化染色。结果 本组胶质瘤MMP 2与integrinβ1表达率分别为 4 3.5 %和 5 6 .5 %。MMP 2与integrinβ1的表达均与胶质瘤的恶性程度有关 ,本组胶质瘤MMP 2的表达与integrinβ1的表达之间有明显的相关性 ,在integrinβ1高表达者中 ,其MMP 2高表达组表达率高于低表达组 (χ2 =5 .4 78,P <0 .0 31)。结论 MMP 2和integrinβ1与胶质瘤侵袭性有关 ,并且两者之间的活动可能有内在联系。

hMLH1、hMSH2、hMSH6、Integrin β1和Ki-67在结直肠癌组织表达对预后的影响分析

目的探究分析错配修复基因(mismatch repair gene, MMR)蛋白中的h MLH1、h MSH2、h MSH6以及整合素β1(Integrinβ1)和Ki-67在结直肠癌组织表达水平以及对预后的影响.方法收集来我院治疗的结直肠癌患者的肿瘤标本162例,采用免疫组织化学染色法测定标本中hMLH1、hM SH2、hM SH6、Integrinβ1和Ki-67的表达水平,分析其影响因素,进行统计学分析比较.结果 (1)162例标本中有36例标本存在hMLH1、hMSH2、hMSH6的表达缺失,缺失率为22.22%;(2)MMR蛋白表达缺失在肿瘤直径(P=0.0005)、Dukes分期(P=0.0248)、肿瘤家族史(P=0.0042)和淋巴结转移(P=0.0014)等方面差异具有统计学意义(P<0.05);(3)Integrinβ1阳性表达水平在Dukes分期(P=0.0002)方面差异存在统计学意义(P <0.05),Ki-67的阳性表达水平在Dukes分期(P=0.0002)、淋巴结转移(P=0.0111)等方面差异具有统计学意义(P <0.05);(4)MMR蛋白表达缺失、Integrinβ1阳性表达和Ki-67的阳性表达与Dukes分期(P=0.006)、淋巴结转移(P=0.023)有关,差异具有统计学意义(P <0.05);(5)dMMR组患者5年的生存率为88.89%,pMMR组患者5年的生存率为59.52%,差异具有统计学意义(P=0.0010); Integrinβ1阳性表达组患者5年生存率为59.69%,阴性表达组患者5年生存率为90.91%,差异具有统计学意义(P=0.0007); Ki-67的阳性表达组患者5年生存率为63.27%,阴性表达组患者5年生存率为93.33%,差异具有统计学意义(P=0.0192).结论患者的MMR蛋白、Integrinβ1和Ki-67的表达水平与患者肿瘤的Dukes分期及淋巴结是否转移具有统计学意义上的相关性,且这三种因素均与患者的预后有关.其中, MMR蛋白表达缺失, Integrinβ1和Ki-67的阴性表达的患者预后情况较好,患者的5年生存率较高.

不同分子亚型乳腺浸润性导管癌组织中Integrin β4、S100A4的表达及意义

目的探讨不同分子亚型乳腺浸润性导管癌组织中整合素β4(Integrinβ4)、S100钙结合蛋白A4(S100A4)的表达及意义。方法以2008年2月—2013年1月217例乳腺浸润性导管癌患者为研究对象,免疫组织化学法检测各癌组织中Integrinβ4、S100A4的表达情况,并分析其与分子分型的关系。结果不同分子亚型乳腺浸润性导管癌的肿瘤原发灶、淋巴结转移情况及组织学分级差异具有统计学意义(P<0.05)。Integrinβ4在Luminal A型、Luminal B型(HER2-)、Luminal B型(HER2+)、Erb-B2过表达型、基底样型中的阳性表达率分别为17.39%、5.88%、6.06%、65.96%、67.86%;S100A4阳性率分别为26.09%、29.41%、27.27%、72.34%、71.43%。Integrinβ4蛋白表达在各型中的阳性表达均有显著差异,S100A4蛋白在Luminal A型、Erb-B2过表达型及基底样型中均有显著差异(P<0.05或P<0.01)。结论 Integrinβ4、S100A4在Erb-B2过表达型、基底样型乳腺浸润性导管癌中高度表达,可能与肿瘤细胞浸润和转移有关。

Integrin β_1在维生素C诱导小鼠诱导性多能干细胞向心肌样细胞分化中的作用

目的:探讨整联蛋白β1(Integrinβ1)在维生素C(Vc)诱导小鼠诱导性多能干细胞(induced pluripotent stemcells,iPSCs)向心肌样细胞(cardiomyocytes,CMs)分化中的作用。方法:在立式显微镜下,分别摘取胚胎(12.5 d、14.5 d及16.5 d)BALB/c胎鼠、新生(出生1 d,P1)和成年BALB/c小鼠的心脏,提取总RNA,半定量及实时定量PCR分析心脏发育不同时期Integrinβ1的表达。利用悬滴培养形成拟胚体(embryoid body,EB)的方法体外诱导iPSCs向心肌样细胞分化,诱导过程中分别添加Vc和integrinβ1抑制剂(HMβ1-1)处理,共分为3组:对照组、Vc处理组和Vc+HMβ1-1处理组。诱导分化的第3、5、7天,半定量及实时定量PCR检测Oct4、integrinβ1和心肌特异性因子(α-MHC、MLC2a)。用免疫荧光染色法检测心肌特异性结构蛋白心肌肌钙蛋白I(cTnI)的表达。结果:Inte-grinβ1在心脏胚胎发育时期(E12.5、E14.5、E16.5、P1)的表达递增(P<0.01),成年期表达有所回落(P<0.01)。半定量及实时定量PCR的结果提示,Vc组α-MHC、MLC2a的表达显著高于对照组(P<0.01),加integrinβ1抑制剂HMβ1-1后表达量降低(P<0.01)。跳动EBs计数的结果提示,诱导分化第10、14、18、22、26天,Vc组跳动EBs的百分率显著高于对照组(P<0.01);而Vc+HMβ1-1组跳动EBs的百分率相对于Vc组显著降低(P<0.01)。免疫荧光染色显示,Vc组有较强的cTnI表达,添加integrinβ1抑制后,Vc+HMβ1-1组cTnI的表达明显减弱。结论:Vc可促进iPSCs向心肌细胞分化,其机制可能是通过integrinβ1介导。

Suspension state promotes extravasation of breast tumor cells by increasing integrin β1 expression

Mechanical microenvironment can strongly affect the metastatic efficiency of circulating tumor cells.However,the effect of suspension state on their extravasation and the mechanisms involved are still unclear.To explore the influence of suspension state on extravasation(including adhesion,spreading and transendothelial migration)of breast tumor cells and its relevant molecular mechanism,MDA-MB-231 cells were cultured on poly(2-hydroxyethyl methacrylate)coated 6-well plates to minic the suspension state.Suspension state promoted adhesion,spreading and transendothelial migration of MDA-MB-231 cells to EAhy926 endothelial cells(ECs)monolayer under both the static condition and 0.5 dyne/cm^(2) flow shear stress(FSS).The number of cells adhered to ECs monolayer reached 2.15(static condition,3 d)and 1.67(FSS,3 d)times,and the number of migration reached 10.60 times,respectively,as compared to that in adhesion state.Moreover,MDA-MB-231 cells knockdown of integrin β1 provoked poor adhesion and transendothelial migration,as compared with MDA-MB-231 cells.But it made no difference in cell spreading.Our results implied the increasing expression of integrin β1 induced by suspension culture promoted the adhesion and transendothelial migration of MDA-MB-231 cells,but had no significant influence on their spreading.

Integrin β1在出生前后大鼠中脑黑质的表达

为了检测integrin β1在出生前后大鼠中脑黑质的表达，并探索其在大鼠黑质神经细胞发育过程中的可能作用，本实验采用RT—PCR、Western blot和免疫组织化学染色方法，来测定胚龄（embryonic day，E）E16 d、E18 d、E20 d的健康SD胎鼠及出生后1d（postnatal day1，P1）、P3d、P7d、P14d、P21d的健康仔鼠中脑黑质组织中integrin β1的表达情况。RT—PCR和Western blot结果显示，在检测的各时间点，大鼠中脑黑质integrin β1都有明显表达，且其mRNA和蛋白质水平变化一致。在E16时，integrin β1 mRNA和蛋白质出现高表达，此高表达一直持续到P7时；而至P14时，integrin β1 mRNA和蛋白质表达水平明显降低，且此低水平表达随发育而持续；P21时与P14时相比无明显差异。而免疫组织化学染色结果显示，在各时间点，大鼠中脑黑质一直有大量integrint31阳性细胞存在。在E16～P14各时间点，integrin β1阳性细胞胞体逐渐增大，P21时与P14时相比，阳性细胞胞体大小无明显变化。而单位面积内integrin β1阳性细胞数，在E16-P7各时间点无明显差异，但至P14时，单位面积内integrin β1阳性细胞数则明显降低，且此降低随发育而持续，P21时与P14时相比无明显变化。以上结果表明，在大鼠中脑黑质发育过程中，integrin β1在E16～P7期间持续高表达，而至P14～P21期间，其表达量明显降低。这种表达变化与大鼠发育期间黑质多巴胺（DA）能神经元的自然凋亡时程相一致，提示integrin β1的表达与此期问黑质DA能神经元的凋亡有关。

Identification of integrin β6 gene promoter and analysis of its transcription regulation in colon cancer cells

BACKGROUND The integrinβ6 gene,which is expressed in epithelial cancer,plays a pivotal role in various aspects of cancer progression.The present research for integrinβ6 regulation mainly focuses on the post-transcription and translation related regulation mechanism and its role in tumorigenesis.The mechanisms of how the integrinβ6 gene is regulated transcriptionally,and the promoter and transcription factors responsible for basic transcription of integrinβ6 gene remain unknown.AIM To clone and characterize the integrinβ6 promoter.METHODS Software analysis was used to predict the region of integrinβ6 promoter.Luciferase reporter plasmids,which contained the integrinβ6 promoter,were constructed.Element deletion analysis was performed to identify the location of core promoter and binding sites for transcription factors.RESULTS The regulatory elements for the transcription of the integrinβ6 gene were located between-286 and-85 and contained binding sites for transcription factors such as STAT3 and Ets-1.CONCLUSION For the first time,we found the region ofβ6 core promoter and demonstrated the binding sites for transcription factors such as Ets-1 and STAT3,which are important for integrinβ6 promoter transcription activity.These findings are important for investigating the mechanism of integrinβ6 activation in cancer progression.