变异型CD44分子和癌细胞的侵袭及转移

变异型CD44（CD44v）对癌细胞侵袭及转移有着密切的关系,本文拟从侵袭、转移癌组织中CD44v的表达情况和CD44v在癌细胞侵袭、转移中的机理两个方面,结合国内外有关研究的结果对CD44v和癌细胞侵袭、转移的关系作进一步综述。     

康莱特抑制结肠癌细胞转移的作用及其分子机制研究

目的以康莱特(KLT)、环磷酰胺(CTX)抑制癌细胞LoVo细胞增殖、迁移,观察对骨桥蛋白(OPN)、基质金属蛋白酶-9(MMP-9)和尿激酶型纤溶酶原激活物(uPA)表达的影响。方法培养人结肠癌LoVo细胞,取处于对数生长期细胞悬液200μL(细胞浓度为5×104/mL)接种,加入不同体积浓度的KLT或CTX培养,以MTT法检测细胞毒副作用;以Transwell小室检测细胞迁移能力;以Western blotting法分析OPN、MMP-9和uPA变化。结果 KLT、CTX抑制LoVo细胞增殖呈时间、剂量依赖性,其IC50 KLT为20μL/mL,CTX为2μM。在CTX、KLT组细胞存活率分别为57%、63%,比对照组均显著降低(x2值分别为54.78、45.90,P均<0.05)。CTX+KLT组细胞存活率为38%,比CTX、KLT组显著降低(x2值分别为7.29、12.50,P均<0.05)。细胞迁移力:空白组细胞数为117±3.5,CTX组为45±1.3,KLT组为67±2.1,比对照组明显减少(t值分别为43.12、27.89,P均<0.05)。CTX+KLT组为21±0.9,比CTX、KLT组显著减少(t值分别为33.94、45.02,P均<0.05)。KLT下调LoVo细胞OPN、MMP-9和uPA表达,并呈现剂量依赖性。与空白对照组、KLT组及CTX组比,KLT联合CTX组抑制LoVo细胞OPN、MMP-9和uPA的表达更明显。结论 KLT可能通过下调OPN、MMP-9及uPA表达,抑制LoVo细胞增殖和转移,且与CTX联合具有协同作用。     

巨噬细胞刺激蛋白受体过表达对癌细胞侵袭能力的初步研究

目的研究巨噬细胞刺激蛋白受体(MST1R)过表达对结肠癌细胞侵袭能力的影响。方法将携有野生型 MST1R(wt-MST1R)cDNA 的质粒 pDR2-wt-MST1R 转染入结肠癌细胞株 RKO,挑选稳定转染克隆。以过河实验和趋化运动实验检测二者的移动能力,以基质浸润实验检测浸润能力,以 Western 印迹检测 E-钙粘连蛋白表达的变化。结果转染并高表达 wt-MST1R 后,RKO 细胞的趋化移动能力明显增加(P<0.01)。过河实验中转染组过河时间为(42.50±4.12)h,而未转染组与载体对照组分别为(69.50±2.52)h 与(70.50±3.42)h(P<0.01)。基质浸润实验中转染组47.90±6.82/视野,未转染组与载体对照组分别为25.90±4,56/视野与26.50±5.36/视野(P<0.01)。转染wt-MST1R 后,E-钙粘连蛋白表达降低(P<0.05)。结论 wt-MST1R 高表达可降低 E-钙粘连蛋白表达,降低肿瘤细胞间的黏附性,增加结肠癌细胞株 RKO 的侵袭能力。提示 MST1R 高表达可能是结肠癌的浸润转移机制之一。     

miR-193b对结肠癌细胞侵袭转移的影响及其机制

目的探讨miR-193b对结肠癌细胞侵袭转移能力的影响及其可能的作用机制。方法 RT-PCR检测高转移细胞系SW620、低转移细胞系中SW480 miR-193b的表达,同时设计miR-193b siRNA及miR-193b利用脂质体2000转染入细胞内,并通过RT-PCR检测其转染效率,利用体外划痕及Transwell实验验证转染miR-193b siRNA及miR-193b后对肿瘤细胞的侵袭转移能力的影响,利用Western印迹验证miR-193b对尿激酶型纤溶酶原激活剂(uPA)的影响。结果 RT-PCR结果显示miR-193b在高转移细胞系SW620中表达水平显著低于转移细胞系SW480;RT-PCR检测转染效率的结果显示转染miR-193b-siRNA的SW480细胞中miR-193b的表达较对照显著降低,而在转染mimic-miR-193b的SW620细胞中其表达水平明显升高;体外划痕实验结果显示SW480-miR-193b-siRNA细胞体外迁移能力显著升高,而在转染mimic-miR-193b的SW620细胞中其迁移能力较对照组显著降低;Transwell小室结果显示体外细胞侵袭及迁移实验中过表达miR-193b均可明显抑制SW620细胞的侵袭转移能力,而抑制miR-193b的表达可显著增加SW480侵袭转移潜能;Western印迹显示过表达miR-193b可显著降低SW620细胞中uPA蛋白的表达,抑制miR-193b可明显提高SW480中uPA蛋白的表达水平。结论 miR-193b通过下调uPA可抑制结肠癌细胞的侵袭和转移。     

结肠癌转移的生物学研究

结肠癌转移的生物学研究陶厚权１王瑞年２林言箴２上海第二医科大学１瑞金医院外科２病理教研室上海市２０００２５ＳｕｂｊｅｃｔｈｅａｄｉｎｇｓＣｏｌｏｎｉｃｎｅｏｐｌａｓｍｓ／ｐａｔｈｏｌｏｇｙＮｅｏｐｌａｓｍｓｍｅｔａｓｔａｓｉｓＲｅｖｉｅｗｌｉｔ...     

Fas相关死亡结构域蛋白增强5-氟尿嘧啶抑制结肠癌细胞生长作用的研究

目的 通过体外和体内实验研究结肠癌细胞在稳定转染Fas相关死亡结构域蛋白(FADD)基因后对5-氟尿嘧啶的敏感性,探讨FADD基因与5-氟尿嘧啶联合治疗结肠癌的可行性.方法 ①RT-PCR和Western印迹法检测稳定转染FADD基因的结肠癌细胞SW480/FADD、稳定转染空载体的结肠癌细胞SW480/neo和正常结肠癌细胞SW480中FADD基因的mRNA和蛋白表达水平.②MTT法检测3种细胞对5-氟尿嘧啶的敏感性.TUNEL法和流式双染法检测细胞凋亡率.Western印迹法检测caspase-8和caspase-3的表达.③裸鼠成瘤实验观察瘤体生长曲线,病理学和TUNEL法检测肿瘤细胞凋亡.结果 ①SW480/FADD细胞FADD基因mRNA和蛋白表达水平明显较SW480和SW480/neo增高(P＜0.05).②5-氟尿嘧啶对SW480/FADD的抑制率明显高于SW480和SW480/neo,差异有统计学意义(P＜0.05).③5-氟尿嘧啶(10 mg/L)干预48 h后,SW480/FADD凋亡率达(33.3±4.5)%,与SW480[(13.9±3.2)%]和SW480/neo[(14.1±3.4)%]间差异有统计学意义(P＜0.05).④5-氟尿嘧啶(10 mg/L)干预48 h后,SW480、SW480/neo的procaspase-8和procaspase-3表达比SW480/FADD增高,而cleaved easpase-8和cleaved caspase-3的表达比SW480/FADD降低(P＜0.05).⑤SW480/FADD对5-氟尿嘧啶更加敏感,瘤体体积增加幅度明显小于SW480和SW480/neo,差异有统计学意义(P＜0.05).结论 稳定转染FADD基因可明显增加结肠癌细胞对5-氟尿嘧啶的敏感性,二者联合应用在结肠癌治疗中有潜在的价值.     

肝癥口服液含药血清对人肝癌BEL-7402细胞系侵袭和转移的影响

目的:探讨肝癥口服液含药血清对人肝癌BEL-7402细胞系侵袭和转移的影响及其机制.方法:应用细胞培养技术培养人肝癌细胞株BEL-7402,采用血清药理学的方法,以原发性肝癌患者服用肝癥口服液前后血清处理肝癌细胞,通过Boyden小室模型测定其侵袭力和黏附力,细胞迁移实验测定细胞运动能力,同时观察细胞形态,流式细胞术测定肝癌细胞基质金属蛋白酶2(MMP2),基质金属蛋白酶抑制剂2(TIMP2),整合素β_1(integrinβ_1)和上皮钙黏附素(E-cd)表达的改变.结果:肝癥口服液含药血清能明显抑制肝癌细胞侵袭力(25.79±2.31 vs 49.54±4.32,P<0.05)、黏附力(42.38±3.19 vs 68.67±5.36,P<0.05)及运动能力(34.72±3.43 vs 54.16±5.35,P<0.05).形态学观察发现,服药后血清组细胞形态较圆,伪足数目较少,MMP2阳性表达细胞减少(289.61±25.32 vs 439.28±22.45,P<0.05),TIMP2阳性表达细胞增多(348.75±34.58 vs 250.53±16.48,P<0.05),MMP2/TIMP2比值下降(0.83 vs 1.76,P<0.05);integrinβ_1表达减少(286.05±28.47 vs 389.57±21.23,P<0.05),E-cd的表达则增加(385.57±26.36 vs 230.72±13.41.P<0.05).结论:肝癥口服液含药血清能抑制肝癌BEL-7402细胞侵袭与转移,其机制可能与直接抑制细胞迁移运动,抑制细胞基质溶解相关基因蛋白MMP2表达,促进TIMP2表达;抑制黏附分子integrinβ_1的表达,促进E-cd表达有关.     

百里醌抑制人胰腺癌BxPC-3细胞体外运动和侵袭的研究

背景:胰腺癌是恶性程度最高的消化道肿瘤,目前吉西他滨依赖的化疗对抑制胰腺癌转移的治疗效果欠佳。研究发现百里醌对多种肿瘤细胞具有抑制增殖、促进凋亡的作用。目的:探讨百里醌对人胰腺癌BxP C-3细胞体外运动和侵袭的影响及其作用机制。方法:常规培养人胰腺癌细胞株BxP C-3,加入不同浓度百里醌进行处理。采用Boyden小室法检测细胞体外运动、侵袭情况;蛋白质印迹法检测细胞FAK、Akt蛋白表达和Akt磷酸化水平的改变;免疫荧光技术检测细胞内FAK表达、细胞黏着斑和F-actin的变化。结果:10、25μmol/L百里醌对BxP C-3细胞体外运动的抑制率分别为43.4%、73.8%,对体外侵袭的抑制率分别为60.5%、75.6%,百里醌呈浓度依赖性地抑制胰腺癌BxP C-3细胞的体外运动、侵袭(P0.05)。百里醌能明显下调BxP C-3细胞FAK表达,并抑制细胞磷酸化Akt的激活。百里醌可诱导FAK弥散分布于胞质,明显抑制黏着斑形成和F-actin的聚合集化。结论:百里醌通过抑制FAK/PI3K/Akt通路的信号转导和激酶活性,浓度依赖性地抑制人胰腺癌BxP C-3细胞的体外运动和侵袭。     

槐耳清膏对结肠癌细胞生长和迁移的影响及其作用机制

目的中药槐耳清膏具有抗肿瘤效应,但其对结肠癌的作用及机制尚不清楚。本研究旨在探讨槐耳清膏对人结肠癌细胞生长和迁移的影响及作用机制。 方法体外培养人结肠癌细胞系HCT116,采用MTT法检测槐耳清膏对肿瘤细胞生长的抑制作用;采用Boyden趋化小室检测槐耳清膏对肿瘤细胞迁移能力的影响;采用荧光定量PCR法检测药物对MMP-9表达的影响;应用流式细胞技术检测药物对细胞凋亡和细胞周期的影响。 结果槐耳清膏对HCT116细胞生长有明显的抑制作用,该抑制作用具有药物剂量和时间依赖性。与细胞共培养96小时后,槐耳淸膏的抑制作用最强;与对照组相比,10 mg/mL、20 mg/mL和30 mg/mL槐耳淸膏对肿瘤细胞的抑制作用分别为25%(P＝0.025),42%(P＝0.032)和67%(P＝0.018)。槐耳清膏能明显抑制肿瘤细胞的迁移,11 mg/mL药物的抑制率为63%(P＝0.01);槐耳清膏(8mg/mL和11mg/mL)处理后的肿瘤细胞中MMP-9的表达量下降(2.73±0.32 vs 4.8±0.36,P＝0.02;1.46±0.25 vs 4.8±0.36,P＝0.004);槐耳清膏可阻滞细胞停留在S期,但对细胞凋亡无明显影响。 结论体外试验证实槐耳清膏对结肠癌细胞的生长及迁移能力具有抑制作用,其作用机制可能是阻滞细胞停留在S期和下调MMP-9的表达。     

Clinicopathological characteristics and prognosis of colon cancer with lung metastasis without liver metastasis: A large population-based analysis

Distant metastasis explains the high mortality rate of colon cancer, in which lung metastasis without liver metastasis (LuM) is a rare subtype. This study is aimed to identify risk factors of LuM and LLM (lung metastasis with liver metastasis) from colon cancer, and to analyze the prognosis of patients with LuM by creating a nomogram. Patients’ information were obtained from the Surveillance, Epidemiology, and End Results (SEER) database. Multivariable logistic regression analysis was used to determine the risk factors for LuM and LLM. Prognostic factors for cancer-specific survival (CSS) and overall survival (OS) were identified by multivariate Cox proportional hazards regression and nomogram models were established to predict CSS and OS. Multivariate logistic regression analysis showed that blacks, splenic flexure of colon tumor, tumor size >5 cm, T4, N3, and higher lymph node positive rate were associated with the occurrence of LuM. Meanwhile, age >65 years old, female, splenic flexure of colon, higher lymph node positive rate, and brain metastasis were independent risk factors for CSS. The C-index of the prediction model for CSS was 0.719 (95% CI: 0.691–0.747). In addition, age, primary site, tumor size, differentiation grade, N stage, and bone metastasis were significantly different between LuM and LLM. The nomograms we created were effective in predicting the survival of individuals. Furthermore, patients with LuM and LLM from colon cancer might require different follow-up intervals and examinations.     

E-钙粘蛋白与肺癌侵袭转移

E-钙粘蛋白(E-cadherin, E-cad)是一类介导细胞与细胞间相互黏附、具有维持组织结构完整性和极性的钙依赖性跨膜糖蛋白.较多研究证实E-cad与多种上皮恶性肿瘤的分化、侵袭、转移和预后相关.本文就E-cad的结构功能、在肿瘤侵袭转移中的作用机制、与肺癌的关系及血清可溶性E-cad检测的临床意义作一综述.     

蚌皮素对结肠癌细胞株调控及其受体后信息传导

研究蛙皮素对人结肠癌细胞株的生长调控及受体后的信息传导途径。方法观察蛙皮素对人结肠癌细胞株ｃｌｏｎｅ Ａ细胞生长调控;应用液体闪烁测量法【闪烁记数仪分别测定细胞内三磷酸肌醇及环磷酸腺苷含量;用荧光测定细胞内游离Ｃａ＾２＋浓度;参考Ｔａｋａｉ法测定蛋白激酶Ｃ活性。     

干细胞培养基成球培养法筛选结肠癌干细胞的生物学特性

目的探讨干细胞培养基成球培养法筛选结肠癌干细胞的相关蛋白表达及生物学特性。方法用干细胞培养基成球培养法筛选结肠癌干细胞,Western blot检测干细胞相关蛋白的表达,流式细胞仪检测细胞周期;同时检测干细胞增殖、黏附、耐药及侵袭能力,并摸索结肠癌干细胞最佳冻存条件。结果用干细胞培养基成球培养法成功培养出了结肠癌细胞球,检测发现干细胞相关蛋白表达增强,静止期细胞比例明显升高,细胞增殖速度慢,黏附性、耐药性、侵袭性均强于亲本细胞。结论干细胞培养基成球培养法筛选的细胞球中富集了肿瘤干细胞,为结肠癌干细胞的研究提供了良好的细胞模型。     

Effect of salinomycin on metastasis and invasion of bladder cancer cell line T24

Objective: To explore the effect of salinomycin on the metastasis and invasion of bladder cancer cell line T24 by regulating the related protein expression in the process of epithelialmesenchymal transition(EMT), and to provide experimental basis for the treatment of urological tumors. Methods: The bladder cancer cell line T24 was cultured in vitro. The rat bladder tumor model was established in vivo. The rats were randomized into two groups, among which the rats in the experiment group were given intraperitoneal injection of salinomycin, while the rats in the control group were given intraperitoneal injection of normal saline. The change of tumor cells in the two groups was observed. Transwell was used to detect the cell migration and invasion abilities, Real-time PCR was used to detect the expression of m RNA, while Western-blot was utilized for the determination of the expressions of E-cadherin and vimentin proteins. Results: The metastasis and invasion abilities of serum bladder cancer cell line T24 after salinomycin treatment in the experiment group were significantly reduced when compared with those in the control group, and the tumor metastasis lesions were decreased from an average of 1.59 to 0.6(P0.05). T24 cell proliferation in the experiment group was gradually decreasing. T24 cell proliferation at 48 h was significantly lower than that at 12 h and 24 h(P0.05). T24 cell proliferation at 24 h was significantly lower than that at 12 h(P0.05). T24 cell proliferation at each timing point in the experiment group was significantly lower than that in the control group(P0.05). The serum m RNA level and E-cadherin expression in the tumor tissues in the experiment group were significantly higher than those in the control group, while vimentin expression level was significantly lower than that in the control group(P0.05). Conclusions: Salinomycin can suppress the metastasis and invasion of bladder cancer cells, of which the mechanism is probably associated with the inhibition of EMT of tumor cells.     

Docosahexaenoic acid suppresses arachidonic acid-induced proliferation of LS-174T human colon carcinoma cells

AIM： To investigate the impact of arachidonic acid （AA） and docosahexaenoic acid （DHA） and their combination on colon cancer cell growth. METHODS： The LS-174T colon cancer cell line was used to study the role of the prostaglandin precursor AA and the omega-3 polyunsaturated fatty acid DHA on cell growth. Cell viability was assessed in XTT assays. For analysis of cell cycle and cell death, flow cytometry and DAPI staining were applied. Expression of cyclooxygenase-2 （COX-2）, p21 and bcl-2 in ceils incubated with AA or DHA was examined by real-time RT-PCR. Prostaglandin E2 （PGE2） generation in the presence of AA and DHA was measured using a PGE2- ELISA. RESULTS： AA increased cell growth, whereas DHA reduced viability of LS 174T cells in a time- and dosedependent manner. Furthermore, DHA down- regulated mRNA of bcl-2 and up-regulated p21. Interestingly, DHA was able to suppress AA-induced cell proliferation and significantly lowered AA-derived PGE2 formation. DHA also down-regulated COX-2 expression. In addition to the effect on PGE2 formation, DHA directly reduced PGE2-induced cell proliferation in a dosedependent manner. CONCLUSION： These results suggest that DHA can inhibit the pro-proliferative effect of abundant AA or PGE2.     

Solitary adrenal metastasis in a patient with sigmoid colon cancer; report of a case

A 73-year-old man had sigmoidectomy for sigmoid colon cancer in December 2001. Although he was followed regularly with chemotherapy, his serum carcinoembryonic antigen (CEA) increased on August 2002. Abdominal computed tomography and magnetic resonance imaging showed a right adrenal mass and no other abnormality. The preoperative diagnosis was a solitary adrenal metastasis from sigmoid colon cancer; the lesion was removed in September 2002. On pathology, adrenal metastasis was confirmed. Although the patient’s serum CEA normalized soon thereafter, 12 months after adrenalectomy, the CEA again increased; the patient had local recurrence of the resected adrenal lesion and liver metastasis. Therefore, the patient was given systemic chemotherapy, but his condition deteriorated, and he died 38 months after adrenalectomy. Adrenal metastasis from colorectal cancer is not unusual; however, a solitary metastasis is rarely found and resected surgically. As surgical treatment of the metastatic lesion could improve patients’ prognosis to some extent if it is detected early, the possibility of adrenal metastasis should be kept in mind when colorectal cancer patients are followed.     

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIM: To investigate the effect and mechanism of action of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS: Human colorectal cancer cell line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h. Nitric oxide (NO) production was measured with Griess reagent. The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane (Matrigel). RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor metalloproteinase-2 (TIMP-2). RESULTS: L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L-NAME, respectively, the ability of the L-NAME treated SL-174T cells to invade the reconstituted basement membrane decreased significantly (t = 8.056, P<0.05; t= 14.467, P<0.01; t= 27.785, P<0.01; and t= 29.405, P<0.01, respectively) and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively (t = 15.116, P<0.01). In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased (t = 71.238,P<0.01) and that of TIMP-2 mRNA was markedly increased (t=-13.020,P<0.01). CONCLUSION: L-NAME exerts anti-invasive and anti-metastatic effects on SL-174T cell line via downregulating MMP-2 mRNA expression and upregulating TIMP-2 mRNA expression.     

尿激酶型纤溶酶原激活物抑制剂1参与非小细胞肺癌的侵袭与转移

目的：探讨尿激酶型纤溶酶原激活物抑制剂1(PAI-1)的表达与非小细胞肺癌(NSCLC)的浸润转移的关系。方法应用免疫组化的方法,观察68例 NSCLC 患者标本中 PAI-1在肺癌组织中的表达及细胞定位,分析其与肺癌病理类型、分期、转移的关系。检测 PAI-1在 NSCLC 细胞系中的表达,分析其与转移相关分子基质金属蛋白酶9的关系,观察细胞因子复合物 Cytomix(TGF-β1+IL-1β+IFN-γ)刺激 A549细胞后 PAI-1的变化及 PAI-1 siRNA 转染对其侵袭能力的影响。结果在 NSCLC 组织标本中,PAI-1的表达主要分布于细胞胞浆,部分可为细胞膜或细胞核,表达可见于肿瘤细胞、间质成纤维细胞、血管内皮细胞及部分巨噬细胞,阳性表达率分别为64.7%、44.1%、25.0%、22.1%(P <0.01)。PAI-1高表达多见于Ⅱ~Ⅲ期、有远处转移的肺癌患者。PAI-1在不同 NSCLC 细胞系具有不同的表达水平,且与转移相关蛋白基质金属蛋白酶9的表达一致。Cytomix 刺激可导致 PAI-1表达的上调,使A549细胞转分化为具有侵袭性的梭形细胞,而给予 PAI-1 siRNA 能抑制该表型的出现。结论 PAI-1在肺癌组织中高表达,提示其在肿瘤发生过程中发挥一定的作用,其不同的表达模式提示其不同的功能需要,PAI-1的表达与转移密切相关。