Effects of 13 T Static Magnetic Fields (SMF) in the Cell Cycle Distribution and Cell Viability in Immortalized Hamster Cells and Human Primary Fibroblasts Cells

Magnetic resonance image （MRI） systems with a much higher magnetic flux density were developed and applied for potential use in medical diagnostic. Recently, much attention has been paid to the biological effects of static, strong magnetic fields （SMF）. With the 13 T SMF facility in the Institute of Plasma Physics, Chinese Academy of Sciences, the present study focused on the cellular effects of the SMF with 13 T on the cell viability and the cell cycle distribution in immortalized hamster cells, such as human-hamster hybrid （AL） cells, Chinese hamster ovary （CHO） cells, DNA double-strand break repair deficient mutant （XRS-5） cells, and human primary skin fibroblasts （AG1522） cells. It was found that the exposure of 13 T SMF had less effect on the colony formation in either nonsynchronized or synchronized AL cells. Moreover, as compared to non-exposed groups, there were slight differences in the cell cycle distribution no matter in either synchronized or nonsynchronized immortalized hamster ceils after exposure to 13 T SMF. However, it should be noted that the percentage of exposed AG1522 cells at G0/G1 phase was decreased by 10% as compared to the controls. Our data indicated that although 13 T SMF had minimal effects in immortalized hamster cells, the cell cycle distribution was slightly modified by SMF in human primary fibroblasts.

Detection of H.pylori DNA in gastric epithelial cells by in situ hybridization

AIM: To investigate the presence of H.pylori DNA within gastric epithelial cells in patients with H.pylori infection and its possible carcinogenic mechanism. METHODS: Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were H.pylori negative and 84 H.pylori positive. H.pylori DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization. RESULTS: In the H.pylori positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the nucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the H.pylori negative group, only one patient with gastric cancer was found H.pylori DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed H.pylori DNA in the cytoplasm of gastric epithelial cells. Furthermore, H.pylori DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells. CONCLUSION: H.pylori DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of H.pylori DNA presence in the nucleus of gastric epithelial cells.

Detection of Human Papilloma Virus Type 16 E6 mRNA in Laryngeal Squamous Cell Carcinoma by In Situ Hybridization

Objective：Laryngeal squamous cell carcinoma（LSCC） is a common malignant tumor in Northeast China and is frequently associated with well-established risk factors like smoking and alcohol abuse.Human papilloma virus（HPV） is an epitheliotropic oncogenic virus that has been detected in a variety of head and neck tumors including LSCC.This retrospective study was to investigate the prevalence of HPV infection in patients with LSCC.Methods：In situ hybridization was performed in 99 patients with LSCC to detect the expression of HPV-16 E6 mRNA.Results：The positive rate of HPV16 E6 mRNA was 36.36%（36/99） in laryngeal squamous cell carcinoma（LSCC）,whereas only 3 of 50（6%） specimens of the normal laryngeal mucosa as a control group showed positive results（P0.05）.Additionally,there was no corelation between HPV16 and age,gender,clinical stage,nodal status and tumor site（P0.05）.Conclusion：The results suggest that the increased prevalence of HPV infection compared to normal laryngeal mucosa and the fact that high-risk HPV types（especially type 16） were the most frequently identified do not allow the exclusion of HPV as a risk factor in laryngeal squamous cell carcinoma.However,their clinical value remains to be further investigated.

Evidence of human papilloma virus infection and its epidemiology in esophageal squamous cell carcinoma

AIM： To look for the evidence of human papilloma virus （HPV） infection in esophageal squamous cell carcinomas （ESCC） and to investigate the potential role and epidemiology of HPV infection in the pathogenesis of esophageal carcinomas in Henan emigrants. METHODS： Papilloma virus （PV） and HPV were determined by UltrasensiveTM S-P immunohistochemistry （IHC） and in situ hybridization （ISH） in esophageal carcinoma tissues （82 cases） and the normal mucosa （40 cases）. RESULTS： IHC revealed that the positive rate of PV was 75.0%, 68.18% and 72.5% respectively while the HPV （16/18-E6） positive rate was 45.0%, 36.36%, 37.5%, respectively in esophageal carcinoma tissue specimens from Henan emigrants,the local citizens and patients in Hubei Cancer Hospital. The PV and HPV （16/18-E6） were negative in all normal esophageal mucosa specimens. No correlation was found between HPV in esophageal squamous cell carcinoma tissues and in grade 1-3 esophageal squamous cell carcinoma cells. In situ hybridization showed that the HPV （16/18） DNA positive rate was 30.0%, 31.8%, 25.0%, respectively in the 3 groups of samples. No positive hybridization signal was found in 40 normal esophageal mucosa specimens. The positive rate of HPV （16/18） DNA in the esophageal carcinoma specimens was significantly higher than that in normal mucosa specimens （P〈0.05）. The positive rate was not different among the 3 groups of esophageal carcinoma tissue specimens （P〉0.05）.CONCLUSION： HPV infection is high in esophageal carcinoma of Henan emigrants, local residents and patients in Hubei Cancer Hospital. HPV is closely related with esophageal squarnous cell carcinoma. HPV infection may play an important role in esophageal squarnous cell carcinoma.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines

AIM: The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. METHODS: Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. RESULTS: Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. CONCLUSION: The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

Expression of exon 13 from the Ki-67 gene in human cells and tissues by digoxigenin-labelled mRNA in situ hybridization

OBJECTIVE: To get insight on the regulatory mechanism of Ki-67 gene expression in malignant cell cycle. METHODS: Non-radioactive in situ hybridization (ISH) was undertaken, combined with immunohistochemistry to study the Ki-67 gene transcription and translation in various human cells and tissues. HeLa cells and fresh colon cancer cells, tonsil, normal pancreas and pancreatic cancer tissues were used in this study. A 435 bp cDNA fragment located in exon 13 of the Ki-67 antigen gene was amplified by polymerase chain reaction (PCR). Digoxigenin-labelled antisense and sense RNA probes were prepared for detecting Ki-67 mRNA, combined with MIB-1 immunohistochemistry. RESULTS: Successful localization of Ki-67 mRNA in human HeLa cells, colon cancer cells, tissues specimen of the tonsil and pancreatic cancer tissue sections was accomplished by digoxigenin-labelling in situ hybridization technique. ISH to colon cancer cells and pancreatic cancer tissue slides showed that much stronger cytoplasm and perinuclear mRNA signals of the Ki-67 gene were present in malignant cells than in normal cells, which was in accordance with MIB-1 nuclear protein signals. CONCLUSIONS: A sensitive and practical in situ hybridization method for the analysis of Ki-67 antigen mRNA in human cell and tissue was developed. Abnormal transcription of exon 13 of Ki-67 gene might be responsible for malignant cell proliferation in colon and pancreatic cancer.

Effect of Matrine on Human Ether à go-go Related Gene (HERG) Channels Expressed in Chinese Hamster Ovary Cells

Objective： To observe the effect of matrine on human ether à go-go related gene （HERG） potassium channels expressed in Chinese hamster ovary （CHO） cells and investigate whether HERG channel is a new target of the pharmacological effect of matrine on arrhythmia and tumor. Methods： HERG channel potassium current in CHO cell was recorded using whole-cell patch-clamp technique, and the influence of matrine on the current was explored. Results： Matrine inhibited HERG potassium current in a dose-dependent manner, and the 50% inhibitory concentration （IC50） was 411±23 μmol/L. Matrine had no significant effect on the activation kinetics, and mainly blocked HERG channels in their closed state. Conclusions： The blocking effect of matrine on HERG channels might be one of the mechanisms against arrythmias and tumors. Unlike most other blockers exerting blocking effect at the intracellular sites by entering the cell with the opening of HERG channel, matrine blocked HERG channels at the extracellular sites.

In situ hybridization of neuropeptide Y gene expression in the endothelial cells of human umbilical blood vessels

In recent years, the endothelial cell has become the popular subject of many scientificresearch fields such as cardiovascular and cancer pathology. The results reported up to nowindicate that the endothelial cell has the complex funtion in synthesis and metabolism, andtherefore plays an important role in the regulation of vascular function. Burnstock’s labfirst observed that many kinds of vasoactive substances are localized in the endothelial

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.

Studies on mechanism of Sialy Lewis-X antigen in liver metastases of human colorectal carcinoma

INTRODUCTIONSialyl Lewis-X antigen ,correlated with carcinoma, is a group of carbohydrate antigen containing oligosaccharide expressed of embryonic tisue and glycoproteins on cell surface of embryonic tissue[1].The SLeX antigen located on cell surface is synthesized principally by two enzymes ,al ,3fucosyltransfrease and a2, 3sialyctransferase.In adults ,SLeX antigen is expressed principally on the surfaces of granulocytic cells and some tumor cells .

Aging-independent and size-dependent genotoxic response induced by titanium dioxide nanoparticles in mammalian cells

Titanium dioxide nanoparticles (TiO2 NPs) are subjected to various transformation processes (chemical,physical and biological processes) in the environment,potentially affecting their bioavailability and toxic properties.However,the size variation of TiO2 NPs during aging process and subsequent effects in mammalian cells are largely unknown.The aim of this study was to illustrate the adverse effects of TiO2 NPs in different sizes (5,15 and <100 nm) during aging process on human-hamster hybrid (AL) cells.There was an aging-time dependent enhancement of average hydrodynamic size in TiO2 NPs stock suspensions.The cytotoxicity of fresh TiO2 NPs increased in a size-dependent manner;in contrast,their genotoxicity decreased with the increasing sizes of NPs.No significant toxicity difference was observed in cells exposed to either fresh or 60 day-aged TiO2 NPs.Both Fresh and aged TiO2 NPs efficiently induced mitochondrial dysfunction and activated Caspase-3/7 in a size-dependent manner.Using mitochondrial-DNA deficient (ρ°) AL cells,we further discovered that mitochondrial dysfunction made significant contribution to the size-dependent toxicity induced by TiO2 NPs during the aging process.Taken together,our data indicated that TiO2 NPs could significantly induced the cytotoxicity and genotoxicity in an aging time-independent and size-dependent manner,which were triggered by mitochondrial dysfunction.Our study suggested the necessity to include size as an additional parameter for the cautious monitoring of TiO2 NPs disposal before entering the environment.

流行性出血热地鼠肾细胞双价灭活疫苗的人体观察

为了研制一种对两型流行性出血热(EHF)均有良好预防效果的灭活疫苗,采用地鼠肾细胞(GHKC)传代适应滴度高(≥8.5logTCID50/ml)的两型EHF病毒株(家鼠型L99株及野鼠型JR株)试制出GHKC双价灭活疫苗(90-1批液体苗和90-2批冻干苗),经卫生部批准,进行了约200人的人体免疫观察。疫苗用福尔马林灭话,加AI(OH)_3佐剂。共免疫242人,其中206人免疫前血清抗体反应阴性。全部接种者除极少数有轻、中度反应,余均无不良反应。两批疫苗接种后,IFA抗体阳转率分别为95.9％及89.2％,ELISA抗体分别为93.5％及91.8％。抽样检查中和抗体反应(每批疫苗30人),用酶斑减少中和试验(EFRNT)检查,两批疫苗对L99株病毒中和抗体阳转率均为100％;对JR株抗体阳转率分别为88.2％及66.7％。每个接种者免前及第一针疫苗接种后56,180,360天血清同时用ELISA法检查抗体,并抽样(每批疫苗30例)检查中和抗体,发现两种抗体的阳性率有逐渐降低的动态变化。两批疫苗三种免疫程序(0,7,28天,0,28,42天及0,28天)接种者的免疫反应基本相似,仅0,28天接种者的抗体水平稍低。对68例接种者于免疫后1年用同一疫苗(90-2批)加强免疫一次,其ELISA及中和抗体均显著升高。试用淋巴细胞转化试验检查20人按种后细胞免疫反应。

人乳头状瘤病毒16在食管癌不同人群中的检出率

背景与目的中国河南省安阳地区是食管癌高发区,有研究发现人乳头状瘤病毒(humanpapillomavirus,HPV)的感染是安阳地区食管癌的重要发病因素。本文研究HPV16型在中国北方不同地区食管癌患者中的感染率及其表达水平,以进一步明确HPV16与食管癌发病的相关性。方法用地高辛标记的HPV16E6探针做原位杂交(insituhybridization,ISH),检测河南省安阳食管癌高发区的食管癌患者(43例)、北京肿瘤医院散发食管癌患者(43例)和内蒙古自治区蒙古族食管癌患者(33例)的食管癌组织中HPV16的感染情况。结果ISH检查结果显示安阳、北京、内蒙古3个地区食管癌患者HPV16感染率分别为81.4%(35/43)、69.8%(30/43)和63.6%(21/33);安阳食管癌患者HPV16感染水平明显高于北京(H=3.91,P<0.05)和内蒙古(H=4.22,P<0.05)的食管癌患者。安阳食管癌患者中HPV16表达呈阳性、强阳性的比例明显高于北京和内蒙古食管癌患者(H=3.95,P<0.05)。结论HPV16在不同地区的食管癌患者中均有较高感染率。安阳食管癌高发区的患者感染HPV16的程度较严重。

女性生殖道人乳头瘤病毒感染与宫颈癌前病变和宫颈癌的关系

目的：探讨女性生殖道人乳头瘤病毒感染与宫颈癌前病变的关系。方法：对２５３例妇科感染患者用ＰＣＲ方法检测ＨＰＶ分型，同时对７８例不同宫颈病变组织行ＨＰＶ１６、１８型原位杂交，并在邻近切片用免疫组化方法检测增殖细胞核抗原。结果：１）女性生殖道ＨＰＶ感染率，由慢性宫颈炎→假性湿疣→疣样病变→尖锐湿疣→ＣＩＮ→宫颈癌，随宫颈病变程度的加重而逐渐升高，ＩＳＨ阳性杂交信号的分布与ＨＥ染色中挖空细胞的分布相一致。２）ＨＰＶ１６、１８型感染率及ＰＣＮＡ表达率均随宫颈病变程度的加重呈升高趋势。挖空细胞核呈ＰＣＮＡ阳性反应，与ＩＳＨ阳性杂交信号出现的部位相一致。结论：ＨＰＶ感染可使宫颈上皮细胞获得较高的增殖活性，也可能通过促进细胞的过度增殖活性而致癌变。

肺腺癌多药耐药细胞特异表达基因的克隆与鉴定

目的：克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法：将肺腺癌多药耐药细胞（ SPC- A- 1/CDDP）作为实验方，肺腺癌细胞（ SPC- A- 1）作为对照方，应用抑制消减杂交技术，构建实验方特异表达 cDNA消减文库；用斑点杂交法初步筛选 cDNA消减文库后，将获得的阳性克隆进行测序和同源性分析（ Genbank），对新的 cDNA序列进行 Northern blot杂交验证。结果：建立了一个肺腺癌多药耐药细胞（ SPC- A- 1/CDDP）特异表达 cDNA消减文库，斑点杂交法初步筛选显示 23个克隆中有 SPC- A- 1/CDDP特异表达 cDNA片断，测序和同源性分析表明 2个 cDNA片断为新序列，其余 cDNA片断与已知基因有 93％～ 100％的同源性， Northern blot杂交结果表明 2个新的 cDNA片断来自 SPC- A- 1/CDDP细胞。结论： 2个新的 cDNA序列可能为未知肺腺癌多药耐药相关基因序列；抑制消减杂交法是克隆特异表达基因的有效方法。

呋喃骈呋喃木脂素的体外抗肿瘤活性

利用 MTT快速比色法 ,以长春新碱 (Vincristin Sulfas)为阳性对照 ,研究了从玄参科(Scrophulariaceae)兰石草属兰石草 (L ancea tibetica Hook Fet Thoms)中提取的呋喃骈呋喃木脂素对人肝癌细胞 (SMMC- 772 1) ,人宫颈癌细胞 (Hela) ,中国仓鼠肺成纤维细胞 (V79)及小鼠黑色素肉瘤细胞 (B16 )的体外抗肿瘤活性 .结果显示 :呋喃骈呋喃木脂素 L C1对 4种细胞均有较强的抑制作用 ,IC50 在 4 0 .2～ 87.9μg/ m L范围 ,它对 B16的抑制作用强于长春新碱 .但是 ,L C2和 L C3均无抗癌活性 (>4 0 0 μg/ m L) ,可能 L C2和 L C3的酚羟基上的 H被糖基取代而使抗癌活性丧失 .

应用抑制消减杂交(SSH)克隆肺腺癌多药耐药细胞特异表达基因

目的 克隆和筛选肺腺癌多药耐药细胞特异表达基因。方法 将肺腺癌多药耐药细胞 (SPC A 1/CDDP)作为实验组 ,肺腺癌细胞 (SPC A 1)作为对照组 ,应用抑制消减杂交技术 ,构建实验组特异表达cDNA消减文库 ;用斑点杂交初步筛选cDNA消减文库后 ,将获得的阳性克隆进行测序和同源性分析 (Genebank)。结果 建立了一个肺腺癌多药耐药细胞 (SPC A 1/CDDP)特异表达cDNA消减文库 ,斑点杂交初步筛选显示 2 3个克隆中有SPC A 1/CDDP特异表达cDNA片断 ,测序和同源性分析表明 2个cDNA片断为新序列 ,其余cDNA片断与已知基因有 96%～ 10 0 %的同源性。结论 2个新的cDNA序列可能为未知肺腺癌多药耐药相关基因序列 ;抑制消减杂交是克隆特异表达基因的有效方法。

16/18型HPV和IMP3在宫颈脱落细胞中的表达和意义

目的观察16/18型人乳头瘤病毒(HPV)和IMP3蛋白在宫颈液基薄层脱落细胞(LCT)中的表达,探讨其在宫颈癌筛查中的价值和意义。方法用原位杂交法检测163例宫颈细胞块中16/18型HPV DNA的表达情况,采用免疫组化法检测IMP3表达情况,并探讨其与宫颈病变程度的相关性。结果在不同细胞学类型病变中16/18型HPV阳性率不同,分别为NILM 10.00%、ASCUS 29.27%、LSIL 52.63%、HSIL 91.43%、SCC 100.00%;IMP3阳性率分别为NILM 2.50%、ASCUS 21.73%、LSIL 44.74%、HSIL 80.00%、SCC 88.89%。NILM、LSIL与HSIL组两两比较,二者的表达在组间均有显著差异(均P<0.05);且16/18型HPV及IMP3的表达均与宫颈病变程度呈正相关,随病变的级别增高而增高,在SCC中阳性率最高。结论 LCT行细胞块制作、联合检测16/18型HPV和IMP3的表达,可提高宫颈癌筛查的准确性,有效分流不同级别的患者,为指导临床治疗及评估预后奠定基础。

EB病毒潜伏感染在不同部位T细胞淋巴瘤中的表达

目的：探讨不同部位Ｔ细胞淋巴瘤（ＴＭＬ）与ＥＢ病毒（ＥＢＶ）感染的关系。方法：对１００例不同部位的ＴＭＬ（结内４０例、结外６０例），采用原位杂交法检测肿瘤细胞ＥＢＶ编码的ＲＮＡ（ＥＢＥＲ１／２）。结果：（１）１００例中ＥＢＥＲ１／２检出率４８％（４８／１００），结内ＴＭＬ检出率３０％（１２／４０），结外ＴＭＬ检出率６０％（３６／６０），结内、结外ＥＢＶ检出率差异有非常显著意义（Ｐ＜０．０１）。（２）结外呼吸道ＴＭＬＥＢＥＲ１／２检出率鼻腔、鼻咽、口咽和肺分别是８８．９％（１６／１８）、７１．４％（５／７）、４７．６％（１０／２１）、３３．３％（１／３）。（３）胃肠道、软组织ＴＭＬＥＢＥＲ１／２检出率分别是１６．７％（１／６）、３３．３％（１／３）。结论：ＥＢＶ感染在不同部位ＴＭＬ中的表达具有明显部位限制性，特别是鼻腔、鼻咽的ＴＭＬ与ＥＢＶ关系最密切。