AIM To explore the components and the distributions of the cytoskeleton network in neoplastic Hep G2 cellsextracted with triton X-100 and (NH4)2SO4.METHODS Using the mouse lung adenocarcinoma cell sublines (C6/C7) with low and high metastasis as acontrol, the human hepatocellular carcinoma cell line (Hep G2) as well as the cell sublines (C6/C7) wasextracted with triton X-100 and/or (NH4)2SO4, then stained with Coomasie blue R250 or labeled withimmunoenzymatic technique to identify the cytokeratin-type or vimentin-type intermediate filamentcomponents and study the distributions of cytoskeleton comparatively.RESULTS Extracted with triton X-100 and/or (NH4)2SO4, then stained with Coomasie blue R250, the cells'cytoskeleton network were showed clearly; still it was very difficult to identify the variations of thecytoskeleton network in morphology by light microscopy when the same cells was extracted with the differentextraction above; compared with the low metastasis cells (C7), most of the high metastasis cells (C6) werelikely showed that the distribution of the cytoskeleton network was more irregular and uneven as well asgathering on one side to the cell neucleus, and so did a few of Hep G2 cells (the percentage of regular andeven distribution of cytoskeleton, C6: 8.0±1.0; C7: 84.0±2.0; Hep G2: 96.0±2.0; n = 500; x2-test,P<0.01). Moreover, extracted with triton X-100 and (NH4)2SO4, then labeled by immunoenzymatictechnique, the mouse lung adenocarcinoma sublines (C6/C7) were positive for cytokeratin antibody only, butthe hepatocellular carcinoma cell (Hep G2) was positive for both cytokeratin and vimentin antibodies.Besides these, in the same cells, the distribution of the intermediate filament network showed by theimmunoenzymatic technique was nearly keeping with that of the cytoskeleton network showed by Coomasieblue R250 stain.CONCLUSION ① It is very difficult to identify the variations of the cytoskeleton network in morphologyby light microscopy when the same cell was extracted with triton X-100 and/or (NH4)2SO4 then stained withCoomasie blue R250 in comparsion. ② The characterizing distribution of the intermediate filament as well asthe ctoskeleton network that was irregular, uneven and gathering on one side to the nucleus in neoplastic cellmight provide a valuable information for studing tumor metastasis. ③ In analysing the components ofintermediate filament protein of malignant tumor cells, the heterogenous proteins (co-expression) must betaken into consideration.

AIM To study the detection of micrometastasis in peripheral blood of patients with gastric carcinomas andits clinical significance.METHODS A cytokeratin 19 (CK19)-specific nested reverse transcriptase-polimerase chain reaction (RT-PCR) assay was developed to detect CK19 expressing cancer cells, the sensitivity was determined by serialdilution method using CK19 expressing gastric cancer cells, the specificity was assessed by examining 12negative controls and 12 positive controls. Then pre-operative peripheral blood from 42 patients with gastriccancer was detected and the relationship between positive results and biological behavior was studied.RESULTS CK19mRNA was expressed in all the 12 gastric cancer tissues but not in peripheral blood from12 healthy individuals；sensitivity of nested RT-PCR amplification for CK19mRNA was confirmed to be 1/106 by serial dilution method using human gastric cancer line SGC-7901; micrometastases in pre-operativeperipheral blood were detected in 13 (30,9%) patients with gastric carcinomas, the frequency ofmicrometastasis in peripheral blood was significantly correlated with tumor size,depth of invasion and TNMstage (x2 test, P<0.05).CONCLUSION Nested RT-PCR amplification for CK19mRNA is a sensitive and specific method for thedetection of micrometastases in peripheral blood in gastric cancer patients; pre-operative detection ofmicrometastasis in peripheral blood may be helpful in the prediction of tumor progression.

慢性异体肾移植病人肾纤维化组织中MMP-2、TIMP-1、Vimentin、Keratin免疫组化定量分析

目的:探讨慢性异体肾移植病人肾纤维化组织中基质金属蛋白酶-2(MMP-2)、金属蛋白酶抑制物(TIMP-1)、波形蛋白(Vimentin)和角蛋白(Keratin)的表达与肾纤维化间的关系.方法:收集我院18例肾移植后不同程度肾纤维化病人及10例正常肾组织,进行MMP-2、TIMP-1、Vimentin 及Keratin的免疫组化定量检测分析.结果:肾纤维化早期,与正常肾组织对比,抗MMP-2和Keratin呈显较强免疫阳性反应,且量增加;随肾纤维化程度增加,抗MMP-2和 Keratin免疫染色阳性反应减弱,量减少,甚至出现阴性;抗TIMP-1和Vimentin免疫染色阳性增强,并随着纤维化进展而量增加.结论:由肾纤维化组织局部Vimentin阳性与相关细胞产生并分泌的细胞因子以诱导MMP-2、TIMP-1的活性改变在慢性肾移植肾病患者的肾纤维化过程中具有关键性作用.