Girdin蛋白调控恶性胶质瘤细胞凋亡的研究

目的 观察抑制恶性胶质瘤LN229细胞中Girdin蛋白的表达对细胞凋亡的影响,并探讨其分子机制.方法 利用小RNA干扰(siRNA)技术,沉默LN229细胞中Girdin的表达,得到实验组克隆(siGirdin/LN229),阴性对照组命名为scr/LN229.采用Western blot技术检测Girdin、细胞色素C(Cyt-C)及Bad蛋白水平的变化.利用增殖实验、噻唑蓝(MTT)比色法检测LN229细胞的存活率;应用流式细胞术检测线粒体释放的Cyt-C量的变化.建立成瘤模型(每组20只),观察Girdin降表达对移植瘤生长情况的影响.结果 siRNA技术有效沉默了LN229细胞中Girdin的表达.增殖实验显示siGirdin/LN229细胞增殖能力下降(P＜0.05),至第6天时实验组细胞数为(15.08±1.17)×104;对照组为(53.93±6.26)×104.MTT实验发现siGirdin/LN229细胞的存活率明显下降(P＜0.05),第5天时实验组为(323.15±57.01)%;对照组为(640.67±59.66)%.流式检测显示siGirdin/LN229细胞Cyt-C含量增加,平均荧光强度为12531.00±1412.98,对照组为183.67±41.55(P＜0.01).分析Western blot结果显示,siGirdin/LN229细胞Cyt-C、Bad的相对灰度值分别为3.57±0.43和4.78±1.03,均高于对照组Cyt-C、Bad的表达水平(相对灰度值分别为1.13±0.1 1、1.78±0.20).体内实验证实Girdin降表达组肿瘤体积明显小于对照组(P＜0.05),至第7.5周时.siGirdin/LN229组肿瘤体积为(36.42±2.00)mm3,对照组为(262.42±24.12)mm3.结论 Girdin能够调控胶质瘤细胞LN229的凋亡,其调节细胞凋亡的机制可能与Cyt-C从线粒体释放及Bad的表达有关.

Abstract：

Objective To investigate the effects of Girdin on apoptosis of LN229 glioblastoma cells by Girdin small RNA interference (RNAi) technology and the molecular mechanisms. Methods Girdin expression was knocked down in LN229 glioblastoma cells by Girdin small interfering RNA (siRNA). The expression levels of Girdin, cytochrome C (Cyt-C) and Bad proteins in glioblastoma LN229 cells were detected by Western blotting. Stable clones were used to measure cells survival ratio by proliferation assay and methyl thiazol tetrazolium ( MTT) assay. Flow Cytometry was used to examine the content of Cyt-C in siGirdin/LN229 and scr/LN229. 20 male athymic Nu/Nu mice were subcutaneously injected with siGirdin/LN229 or scr/LN229 cells respectively to study the growth of tumor in each group. Results Western blotting showed that the expression of Girdin was decreased significantly. The relative intensity of Cyt-C and Bad in siGirdin/LN229 cells was 3. 57 ±0.43 and 4. 78 ± 1. 03 respectively, and the relative intensity of Cyt-C and Bad in scr/LN229 was 1. 13 ±0. 11,1. 78 ±0.20 respectively (P＜0. 05). The mean fluorescence density of Cyt-C in siGirdin/LN229 and scr/LN229 by flow cytometry was 12 531.00 ±1412. 98 and 183. 67 ±41. 55 respectively. Meanwhile, the number of siGirdin/LN229 cells was obviously decreased as compared with control group in proliferation assay. The number of siGirdin/LN229 cells on the 6th day was (15. 08 ± 1.17) x 104, and that of scr/LN229 cells was (53. 93 ± 6. 26) x 104. MTT assay revealed the survival rate of siGirdin/LN229 on the 5th day was (323. 15 ± 57. 01) % , and that of scr/LN229 was (640.67 ±59.66)%, P＜0.05. Additionally, the volume of xenograft tumors in siGirdin/LN229 group was decreased significantly as compared with control group. The volume of xenograft tumors in siGirdin/LN229 group was (36.42 ±2. 00) mm3, and that in scr/LN229 group was (262. 42 ±24. 12) mm3 at the 7.5th week. Conclusion Girdin plays a critical role in apoptosis of glioblastoma cells. Inhibition of Girdin by siRNA could promote the apoptosis of glioblastoma LN229 cells through the release of Cyt-C from mitochondria and up-regulation of Bad.     

Girdin蛋白调控恶性胶质瘤细胞凋亡的研究

Objective To investigate the effects of Girdin on apoptosis of LN229 glioblastoma cells by Girdin small RNA interference (RNAi) technology and the molecular mechanisms. Methods Girdin expression was knocked down in LN229 glioblastoma cells by Girdin small interfering RNA (siRNA). The expression levels of Girdin, cytochrome C (Cyt-C) and Bad proteins in glioblastoma LN229 cells were detected by Western blotting. Stable clones were used to measure cells survival ratio by proliferation assay and methyl thiazol tetrazolium ( MTT) assay. Flow Cytometry was used to examine the content of Cyt-C in siGirdin/LN229 and scr/LN229. 20 male athymic Nu/Nu mice were subcutaneously injected with siGirdin/LN229 or scr/LN229 cells respectively to study the growth of tumor in each group. Results Western blotting showed that the expression of Girdin was decreased significantly. The relative intensity of Cyt-C and Bad in siGirdin/LN229 cells was 3. 57 ±0.43 and 4. 78 ± 1. 03 respectively, and the relative intensity of Cyt-C and Bad in scr/LN229 was 1. 13 ±0. 11,1. 78 ±0.20 respectively (P＜0. 05). The mean fluorescence density of Cyt-C in siGirdin/LN229 and scr/LN229 by flow cytometry was 12 531.00 ±1412. 98 and 183. 67 ±41. 55 respectively. Meanwhile, the number of siGirdin/LN229 cells was obviously decreased as compared with control group in proliferation assay. The number of siGirdin/LN229 cells on the 6th day was (15. 08 ± 1.17) x 104, and that of scr/LN229 cells was (53. 93 ± 6. 26) x 104. MTT assay revealed the survival rate of siGirdin/LN229 on the 5th day was (323. 15 ± 57. 01) % , and that of scr/LN229 was (640.67 ±59.66)%, P＜0.05. Additionally, the volume of xenograft tumors in siGirdin/LN229 group was decreased significantly as compared with control group. The volume of xenograft tumors in siGirdin/LN229 group was (36.42 ±2. 00) mm3, and that in scr/LN229 group was (262. 42 ±24. 12) mm3 at the 7.5th week. Conclusion Girdin plays a critical role in apoptosis of glioblastoma cells. Inhibition of Girdin by siRNA could promote the apoptosis of glioblastoma LN229 cells through the release of Cyt-C from mitochondria and up-regulation of Bad.     

重组人血管内皮抑制素对小鼠肿瘤和心肌中血管结构及血管生成相关因子表达的影响

目的 观察重组人血管内皮抑制素对小鼠肿瘤及心肌中微血管影响的差异.方法 40只小鼠随机分为空白对照组(未荷瘤,生理盐水100 μl/d)、药物对照组(未荷瘤,重组人血管内皮抑制素400 μg/d)、模型组(荷瘤,生理盐水100 μl/d)和实验组(荷瘤,重组人血管内皮抑制素 400 μg/d),分别处理28 d.实验前后称量小鼠体重,测量移植瘤体积.采用免疫组化法检测小鼠心脏和移植瘤组织中基质金属蛋白酶(MMP)-2、MMP-9、缺氧诱导因子1α(HIF-1α)以及血管内皮生长因子(VEGF)蛋白的表达情况,计数微血管密度(MVD).以CD34与Masson双染法观察微血管结构的变化.结果 实验组小鼠肿瘤体积的增加值为(48.18±37.31)mm3,低于模型组[(113.80±73.27)mm3,P＜0.05];各组小鼠体重变化的差异无统计学意义(P＞0.05).应用重组人血管内皮抑制素后,移植瘤组织中MMP-9和VEGF蛋白的表达显著降低,移植瘤组织中MMP-2、HIF-1α以及心肌组织中MMP-2、MMP-9、HIF-1α和VEGF蛋白的表达均无显著变化;移植瘤组织的MVD显著降低,胶原覆盖血管的比例升高,而心肌组织的MVD和结构几乎无变化.结论 重组人血管内皮抑制素可通过下调移植瘤组织中MMPs和VEGF蛋白的表达,降低MVD,抑制移植瘤的生长,并可使移植瘤血管趋向成熟,但不能降低心肌组织中MMPs的表达和成熟血管的MVD.

Abstract：

Objective To compare the effect of rh-endostatin on micrangium in tumor and myocardial tissue in nude mice. Methods Nude mice were randomized into 4 groups(10 mice in each group), blank control group (without tumor burden, received NS 100 μl·d-1 injection), drug control group (without tumor burden, received rh-endostatin 400 μg·d-1 injection), model group (with tumor burden, received NS 100 μl·d-1 injection) and treatment group (with tumor burden, received rh-endostatin 400 μg·d-1 injection) for 28 days. The tumor volume and body weight of the mice were measured before and after administration. The expression of CD34, MMP-2, MMP-9, HIF-1α and VEGF in the myocardium and tumor were detected by immunohistochemistry. The vascular structure was observed by immunoenzymatic CD34 and Masson double staining. Results The increase of tumor volume of the treatment group[(48.18±37.31) mm3]was significantly lower than that in the model group [(113.80±73.27) mm3). The changes of body weight was not significant different among the four groups. After treated with rh-endostatin, the expressions of MMP-9 and VEGF in tumors were significantly down-regulated, but the expressions of MMP-2 and HIF-1α in the tumor were not. The microvessel density (MVD) in the tumors of treatment group was significantly decreased compared with that of model group. The proportion of tumor vessels covered by collagen in the treatment group was increased compared with that of the model group. However, MVD and micrangium in myocardium were not changed significantly. Conclusion Rh-endostatin can decrease the expression of MMP-9, VEGF and MVD, inhibit the tumor growth and normalize tumor micrvangium in tumor but not weaken the MMPs and MVD of mature micragium in myocadium.     

水通道蛋白4调控恶性胶质瘤细胞凋亡的研究

Objective To investigate the effects of apuaporin 4 (AQP4) on apoposis of LN229 glioblastoma cells by AQP4 small RNA interference (siRNA) technology and the molecular mechanism.Methods AQP4 expression was knocked down in LN229 glioblastoma cells by AQP4 siRNA (scr/LN229 and siAQP4/LN229 cells). The expression level of AQP4 protein in LN229 cells was detected by Western blotting. Stable clones were used to measure cells survival ratio (6 days) by methyl thiazol tetrazolium ( MTT) assay. Flow cytometry (FCM) was used to examine the content of cytochrome C in siAQP4/LN229 and scr/LN229. Western blotting was used to measure the levels of cytochrome C, bcl-2 and Bad. Nu/Nu mice were subcutaneously injected with siAQP4/LN229 and scr/LN229 cells to study the growth of tumor in the two groups (20 mice in each group). Results Western blotting showed that the expression of AQP4 was decreased significantly; MTT assay suggested that the survival ratio was decreased with deficiency of AQP4: the survival ratio of scr/LN229 was (587.00 ±4.68)%, and the ratio of siAQP4/N229 was (317. 00 ± 26. 30)% at sixth day (P＜0. 05). The mean fluorescence density of cytochrome C in scr/LN229 and siAQP4/LN229 by FCM was (57.2 ± 16.64) and (468.8 ±46.9) respectively (P＜0.05). The relative intensity of cytochrome C and Bad in siAQP4/N229 cells was ( 1. 62 ±0. 16) and (1. 30 ±0. 24) respectively, and that in scr/LN229 cells was (0. 83 ±0. 29) and (0. 56 ±0. 21) respectively (P＜0. 05). The intensity of bcl-2 in siAQP4/N229 cells (0.53 ±0.03) was decreased compared to scr/LN229 cells (0. 73 ± 0. 12) ( P ＜ 0. 05). The subcutaneous mouse xenograft model showed that the mean tumor volume of scr/LN229 group was (402. 67 ±34. 27) mm3, and that of siAQP4/N229 group was (65. 15 ±32. 12) mm3 at the 6th week. Conclusion AQP4 can regulate apoptosis of glioblastoma cell line LN229, which may be related with the release of cytocherome C, Bad and bcl-2.     

BRCAl基因突变与年轻患者乳腺癌发生的相关性分析

目的 筛查年轻乳腺癌BRCAl基因的突变位点及SNP携带情况,探讨BRCAl基因突变与年轻乳腺癌发生的关系.方法 来自我院2004年1月-2006年8月收集的乳腺癌组织共30例,其中5例有至少1个一级亲属患乳腺癌,发病年龄≤35岁.由乳腺癌组织提取基因组DNA,对BRCAl基因第2、11C、11F、11L、11I、16、20外显子的编码序列进行PCR扩增.扩增产物进行DNA直接测序证实,利用DNA Star-MagAlign软件进行序列比较.结果 BRCAl基因中共发现14个序列变异,有3个移码突变(cDNA2639、2640delTA、3343delG及3398delT)和11个点突变(cDNA 2570 C>T、cDNA2620 A>T、1473A>G、1561C>T、1594G>A、2206A>G、2227T>C、2659C>A、2806T>C、3307A>G、3375G>A),其中3个乳腺癌家族史阳性.突变率为10%(3/30).第16及20外显子未发现突变.结论 BRCAl突变主要位于第11号外显子上,乳腺癌家族史阳性的年轻乳腺癌突变率高,3个移码突变可能与年轻乳腺癌发生相关.     

CD44v6在乳腺癌患者化疗前后的表达及意义

背景与目的：CD44是粘附分子家族的成员,与肿瘤的形成及转移相关,尤其是变异型亚型CD44v6更是与多种肿瘤的侵袭和转移密切相关。本文旨在研究乳腺癌患者血、癌组织、淋巴结中CD44v6的表达及化疗对CD44v6表达的影响和意义。方法：用免疫组化方法检测癌组织CD44v6的表达,用流式细胞术检测血和淋巴结中CD44v6的表达。结果：乳腺癌组织中淋巴细胞（t=4.71,P＜0．05）和阳性淋巴结（t=5.11,P＜0．05）的CD44v6的表达与对照组相比显著增高,而患者外周血淋巴细胞CD44v6的表达也有所升高,但无统计学意义（t=0.98,P＞0．05）。乳腺癌细胞CD44v6的表达强阳性,且阳性率100％,而良性乳腺组织呈阴性表达。化疗后癌细胞CD44v6的表达仍是100％,血和淋巴结中淋巴细胞CD44v6的表达有所下降,但无意义,只有癌组织中淋巴细胞CD44v6的表达水平显著下降（t=4.13,P＜0．05）。结论：CD44v6的表达与乳腺癌患者的淋巴结转移和预后密切相关,化疗后癌组织中淋巴细胞CD44v6表达明显降低。     

抗肿瘤人源性抗体研究进展

人源性抗体具有免疫原性小、特异性好、亲和力高等优点，可以通过不同的目标靶向、不同的方式作用于肿瘤组织。目前，人源性抗体在肿瘤治疗中起着重要作用。     

非霍奇金淋巴瘤骨髓受累的免疫表型探讨

目的：探讨非霍奇金淋巴瘤（NHL）骨髓受累的免疫表型特点方法：应用流式细胞术对NHL患者的骨髓标本进行检测，收集受累者的CD分子表达数据。结果：1）在46例受检者中，31例阳性，阳性率67.39％，（95％可信区间53．84％，80．94％）经骨髓形态学检查确证为骨髓受累。2）31例阳性者中，B细胞NHL23例，T细胞NHL7例．NK细胞NHL1例B细胞NHL标记抗原出现频率最高的为CD19，CD20；T细胞NHL标记抗原出现频率最高的为CD7。6例B细胞淋巴瘤同时表达T细胞抗原，1例B细胞淋巴瘤还表达髓系抗原标志。结论：1）NHL骨髓受累免疫表型特点为B细胞来源：CD19、CD20；T细胞来源：CD7。2）T、B细胞抗原同时表达不少见．3）可同时表达髓系抗原．     

流式细胞术(CD45/SSC设门法)检测恶性淋巴瘤骨髓受累的临床意义

目的:探讨流式细胞术(CD45/SSC设门法)检测恶性淋巴瘤骨髓受累的临床价值.方法:采用配对计数资料的实验设计,应用流式细胞仪对恶性淋巴瘤患者的骨髓标本进行检测,同时行骨髓涂片检查.结果:对34例恶性淋巴瘤患者的骨髓同时行上述两种方法检测,流式细胞术阳性率67.65%(23/34),95%可信区间(51.92%,83.37%),涂片法阳性率11.76%(4/34),95%可信区间(0.94%,22.58%).经配对计数资料X2检验,差别有统计学意义.结论:流式细胞术是检测恶性淋巴瘤骨髓受累的有效方法,检出率67.65%,优于传统的涂片法.     

晚期非小细胞肺癌循环血管内皮细胞水平的研究

目的分析晚期非小细胞肺癌(NSCLC)患者治疗前后外周血中循环血管内皮细胞(CEC)的变化,探讨CEC在NSCLC中的应用价值。方法采用NP方案联合内皮抑素(治疗组)及单用NP方案(对照组)治疗晚期NSCLC 67例,流式细胞法检测治疗前后外周血CEC数量及细胞角蛋白(CK)水平。结果治疗组临床有效率为44．4％,对照组为27．3％(P=0．176);治疗组临床受益率为80．0％,对照组为50．0％(P=0．012)。治疗组肿瘤进展时间(TTP)为146．7 d,对照组为91．1 d (P=0．061)。当TTP的cut-off值>170时,两组TTP比较,差异有统计学意义(cut-off值=170,P= 0．034;cut-off值=180,P=0．009)。治疗组CEC平均下降(0．29±0．47)％,对照组平均下降(0．01±0．43)％(P=0．033)。全部患者治疗前CEC与CK水平呈正相关(r=0．381,P=0．013),治疗后亦呈正相关(r=0．450,P=0．004)。结论化疗联合血管靶向治疗晚期NSCLC优于单用化疗,CEC是一个较好的预测疗效的指标。