2号、18号染色体微卫星多态性与甲状腺癌的关系

目的 探讨特定位点微卫星DNA序列不稳定性(MSI)及杂合性缺失(LOH)与人甲状腺癌发生、临床病理特征及预后的关系.方法 选取THRA1、D2S123、D11S912、BAT-26四个位点,应用聚合酶链反应(PCR)和变性聚丙烯酰胺凝胶电泳技术,对60例人甲状腺癌患者的MSI及LOH表达情况进行研究.结果 THRA1位点MSI检出频率为43.3%,D2S123为36.7%;甲状腺滤泡癌中D2S123检出率为100%,未检测到BAT-26;D18S58的检出率为26.7%;BAT-26在甲状腺癌中检出率为6.7%;LOH 的检出率分别为33.3%、26.7%、23.3%和16.7%.术后随访5年,MSI/LOH阳性的甲状腺癌较阴性者生存期更长(P＜0.05). 结论 在2号和18号染色体中检测到微卫星阳性率较高;D2S123位点MSI与滤泡型甲状腺癌相关性有统计学意义;D18S58位点MSI的阳性率与高龄患者、晚期肿瘤密切相关;BAT-26在甲状腺癌中检出率最低.MSI/LOH导致基因组不稳定,在甲状腺肿瘤发生过程中发挥作用.MSI/LOH阳性的甲状腺癌患者较阴性者生存期更长.     

老年妇女外阴癌及外阴营养不良与p53突变及人乳头瘤病毒感染的相关性

目的：探讨老年妇女外阴癌组织中p53表达及人乳头瘤病毒（HPV）感染与外阴癌发生及临床发展的关系。方法：应用免疫组化法检测老年妇女外阴鳞状细胞癌（外阴癌）68例及老年妇女外阴营养不良38例病例蜡块包埋组织中p53,HPV的表达情况，并选用15例外阴正常皮肤作为对照。结果：p53,HPV在老年妇女外阴癌组织中阳性表达率分别为54．4％，45．6％，老年妇女外阴营养不良组织中为18．4％，55．3％，老年妇女正常外阴皮肤无阳性表达。外阴癌与外阴营养不良，正常对照比较差异有显著性（分别为P＜0．05及P＜0．01）。结论：老年妇女外阴癌组织中p53,HPV阳性表达率较高。p53突变在老年妇女外阴癌的发生过程中起重要作用，高危型HPV的感染促进外阴癌的发生。     

乳腺癌FHIT基因微卫星不稳定性及其蛋白表达的研究

目的分析乳腺癌脆性三联组氨酸基因（FHIT）微卫星不稳定性发生情况及与FHIT蛋白表达缺失的关系，以探讨乳腺癌发生的机制。方法选取FHIT基因的D3S1234、D3S1300位点，采用PCR、聚丙烯酰胺凝胶电泳、银染法，对66例乳腺癌患者肿瘤组织进行微卫星不稳定性（MSI）检测，用免疫组化方法检测FHIT蛋白的表达情况。结果30．3％（20／66）的乳腺癌组织至少在一个位点出现MSI；59．1％（39／66）的肿瘤组织发生FHIT蛋白表达缺失或明显下降，17例MSI（＋）病例发生FHIT蛋白低水平表达或缺失，MSI（＋）与FHIT蛋白表达缺失有相关性（P〈0．05）。FHIT蛋白表达缺失与肿瘤组织学分级有相关性（P〈0．05）。结论FHIT基因位点的微卫星不稳定性是FHIT蛋白表达下调的可能的分子机制之一，FHIT基因在乳腺癌的发生发展中起重要作用。     

Loss of heterozygosity and microsatellite instabilities of fragile histidine triad gene in gastric carcinoma

AIM: To detect the loss of heterozygosity (LOH) and microsatellite instabilities (MSI) of fragile histidine triad (FHIT) gene in gastric carcinoma and to study their association with the clinical pathological characteristics of gastric carcinoma. METHODS: LOH and MSI of FHIT gene were detected at four microsaterllite loci D3SI3H, D3S4I03, D3SI48I and D3S1234 using PCR in matched normal and cancerous tissues from 50 patients with primary gastric cancer. RESULTS: The average frequency of LOH and MSI of FHIT gene in gastric cancer was 32.4% and 26.4% respectively. LOH and MSI of FHIT gene in gastric cancer had no association with histological, Borrmann, and Lauren's classification. LOH of FHIT gene in gastric cancer was related to invasive depth. The frequency of FHIT LOH in gastric cancer with serosa-penetration was obviously higher than that in gastric cancer without serosa-penetration (73.5% vs 37.5%, P < 0.05). MSI of FHIT gene in gastric cancer was associated with the lymph node metastasis. The frequency of MSI in gastric cancer without lymph node metastasis was significantly higher than that in gastric cancer with lymph node metastasis (66.7% vs 34.3%, P < 0.05). CONCLUSION: LOH of FHIT gene is correlated with invasive depth of gastric carcinoma. MSI of FHIT gene is correlated with lymph node metastases. LOH and MSI of FHIT gene play an important role in carcinogenesis of gastric cancer.     

食管癌组织微卫星DNA序列不稳定性和杂合性缺失及预后的研究

目的探讨微卫星DNA序列不稳定性（MSI）和杂合性缺失（LOH）与人食管癌发生、临床病理特征及预后的关系。方法应用聚合酶链反应（PCR）和变性聚丙烯酰胺凝胶电泳技术,对30例人食管癌中MSI及LOH阳性情况进行研究,术后随访5年,了解预后。结果D3S1067位点MSI发生检出频率较高,为26.7%;D18S58位点MSI阳性率为20%。MSI的发生在食管小细胞癌中较食管鳞癌为高（P〉0.05）;MSI、LOH与肿瘤的病理分级、PTNM分期、有无区域淋巴结转移和浸润深度无关（P〉0.05）。结论食管癌在3p和18q染色体位点均存在微卫星不稳定现象;D3S1067和D18S58二个位点上MSI与食管癌的临床病理类型均相关;研究未发现这两个位点MSI、LOH与食管癌的临床分期、细胞分化程度、癌组织浸润深度和有无区域淋巴结转移等参数相关;3p位点基因的改变在食管鳞癌发生过程中具有较重要意义。     

FHIT基因在肺癌中的缺失与HPV感染相关性的研究

目的　探讨肺癌发生的分子生物学机制 ;方法　采用逆转录 -巢式聚合酶链反应 (reverse- tape polymerase chainreaction)的方法对 4 2例肺癌及 10例正常肺组织中的 FHIT(Fragile histidine triad)基因的缺失情况进行检测 ,并用 PCR技术检测了肺癌组织中人乳头状瘤病毒 (human papilloma virus,HPV)的 DNA片段 ;结果　 6 6 .7% (2 8/ 4 3)肺癌组织中检测到 FHIT基因的缺失 ,而正常组织中未检测到 FHIT基因的缺失 ,二者差别有显著意义 (p<0 .0 1)。 4 2例肺癌组织中有 8例检测到 HPV的片段 ,阳性率为 19% (8/ 4 2 ) ,正常组织中未检测到 HPV的片段 ,且在 8例 HPV阳性的标本中均有 FHIT基因缺失。结论　 FHIT基因的缺失在肺癌的发生中起一定的作用 ;可与肺癌的不同病理分型、分化程度及临床分期无关 ;但与吸烟有一定的相关性 ;同时 HPV的感染与肺癌的发生有一定关系 ,且与 FHIT基因缺失呈正相关     

急性白血病ATM基因和BRCA2基因的杂合性缺失

目的：探讨急性淋巴细胞白血病（ALL）与急性非淋巴细胞白血病（ANLL）ATM基因和BRCA2基因杂合性缺失及其相互关系。方法：应用PCR－变性聚丙烯酰胺凝胶电泳－银染技术检测ATM基因D11S2179位点和BRCA2基因D13S260位点的杂合性缺失（LOH）。结果：64例ALL患者中两位点LOH发生率为18.7%；D11S2179和D11S260两位点LOH的发生频率分别为12.5%和14.0%；两位点同时发生LOH的频率为7.8%。38例ANLL患者中两位点LOH的发生率为7.9%，D11S2179和D13S260两位点的LOH发生频率分别为2.6%和5.2%。两组总发生率比较差异有显著性意义（P＜0.05）。结论：ATM基因和BRCA2基因的LOH可能参与ALL的病理发生，而与ANLL无明显相关性。     

小肠腺癌中微卫星不稳定的特征与意义

目的 检测小肠腺癌组织中微卫星不稳定(MSI)情况,评价其在小肠腺癌中的特征及意义.方法 收集40例小肠腺癌石蜡标本,分离基因组DNA,运用聚合酶链-简单序列长度多态性(PCRSSCP)方法检测MSI情况.结果 40例小肠腺癌中14例表现为1个或1个以上位点不稳定,MSI阳性率为35.0%.其中 8例表现为2个或2个以上位点不稳定,高度MSI(MSI-H)率为20.0%;6例表现为1个位点不稳定,低度MSI(MSI-L)率为15%.其余26例微卫星稳定.小肠腺癌MSI者年龄较小.MSI-H者女性多见,细胞分化程度较好,肿瘤多发生于十二指肠(P＜0.05),40例患者的5个微卫星位点共检出27例次MSI,其中BAT26和D2S123位点的出现频率分别为29.6%(8/27)和33.3%(9/27),与其他3个位点比较差异有统计学意义(P＜0.05).结论 MSI是小肠腺癌中的一个常见分子事件.BAT26和D2S123位点是确定小肠腺癌MSI较敏感的微卫星位点.     

急性白血病p16基因微卫星不稳定性及杂和性缺失的研究

目的分析急性白血病（AL）患者p16基因连锁的微卫星不稳定性（MSI）和杂和性缺失（LOH），了解p16基因改变与AL发生的关系。方法采用多重PCR方法检测53例AL患者骨髓及口腔黏膜细胞标本的p16基因连锁的3个微卫星位点（D9S162、D9S1748、D9S171），观察其MSI及LOH情况。结果53例AL患者中，MSI检出率为43．4％（23／53）；位于9p21的p16基因连锁的微卫星D9S162、D9S1748、D9S171的LOH发生率分别为0（0／53）、5．7％（3／53）和9．4％（5／53），MSI发生率分别为13．2％（7／53）、7．6％（4／53）和7．6％（4／53）。结论AL患者p16基因连锁微卫星均可检测到高频率的MSI和LOH，说明p16基因突变与AL发生、发展有关。     

中国人甲状腺癌D2S123、D18S58、BAT-26基因的遗传不稳定性及意义

目的探讨中国人甲状腺癌特定位点微卫星DNA序列基因遗传不稳定性（MSI）及其临床意义。方法选取D2S123、D18S58、BAT-26三个位点,应用聚合酶链反应（PCR）和变性聚丙烯酰胺凝胶电泳技术,对30例人甲状腺癌中MSI表达情况进行研究。结果D2S123、D18S58、BAT-26位点MSI检出率分别为36.7%。甲状腺滤泡癌中D2S123位点MSI检出率为100%,没有检测到BAT-26位点MSI。D18S58位点MSI的阳性率与高龄患者、晚期肿瘤密切相关。术后随访5a,MSI阳性的甲状腺癌较阴性者生存期更长（P〈0.05）。结论甲状腺癌中存在D2S123、D18S58、BAT-26位点的MSI,但检出率各不相同。上述位点MSI在甲状腺肿瘤发生过程中发挥作用,且对患者预后影响。     

老年人食管鳞状上皮化生和不典型增生及腺癌序列微卫星改变

目的评估老年人食管鳞状上皮和化生-不典型增生-腺癌的微卫星变化。方法应用稀释性聚合酶链反应（PCR）方法检测存档手术切除的食管癌标本中的D2S123、D3S1616、D3S1300、BATRII、D5S346、D17S787和D18S61位点微卫星的变化。结果在非稀释DNA中，17例食管鳞状细胞癌和12例腺癌微卫星不稳定性（MSI）的频率分别是52．9％（9例）和41．7％（5例），杂合性丢失（LOH）的频率分别是23．5％（4例）和16．7％（2例），两者差异均无统计学意义（P〉0．05）。在8例食管鳞状上皮和化生-不典型增生-腺癌组织稀释DNA中，MSI和LOH频繁出现，与其非稀释DNA的结果比较，差异均有统计学意义（P〈0．05）。结论MSI和LOH在上述组织中普遍存在，它们可能是食管腺癌发生、发展的早期事件。     

Lymphatic metastasis and nm23H1 genetic instability in Chinese colon cancer patients

AIM: To investigate the pathogenic mechanism of colon cancer at the molecular level and to elucidate the relationship between intercellular adhesion molecule-1 (ICAM-1) and nm23H1 genes and Chinese patients with colon cancer.METHODS: DNA was extracted from paraffin-embedded materials. Polymerase chain reaction-single s~and conformation polymorphism (PCR-SSCP) was used to analyze MSI and LOH. Expression of ICAM-1 was detected by Envision immuno-histochemistry. Experimental results were analyzed with Leica-Qwin computer imaging techniques and SPSS software of statistics.RESULTS: ICAM-1 expression of lymphatic endothelium was negative in normal colon and posi0ve in colon cancer respectively. The number of lymphatics positive for ICAM-1 was gradually increased with degree of cancer invasion (P&lt;0.01). In the group with metastasis of colon cancer, bhe number of lymphatics positive for ICAM-1 in lymph nodes was more than that in the group with no metastasis (P&lt;0.01). The frequency of MSI, LOH and nm23H1 protein was 26.67%, 20.00% and 53.33% in colon cancer, respectively. In TNM staging, MSI (43.75%) and nm23H1 protein (81.25%) in stages I+II weredetected more easily than the corresponding indexes (MSI:7.14%, P&lt;0.05 and nm23H,: 21.43%, P&lt;0.01) in stages III+IV. By comparison, the frequency of LOH (35.71%) in stages III+IV was more than that of LOH (6.25%, P&lt;0.05) in stages I+II. LOH exhibited a rising trend along with the Duke‘s staging, nm23H1 protein in the group of tubular adenocarcinoma (60.00%) was higher expressed than that in the group of mucoid adenocarcinoma (20.00%) (P&lt;0.01),and exhibited a rising trend with the differentiation degrees of tubular adenocarcinoma, nm23H, protein in MSI positive group was higher expressed (75%) than that in MSI negative group (45.45%, P&lt;0.05).CONCLUSION: The expression of ICAM-1 in lymphatic vessels is beneficial to the judgement of the invasion and metastasis ability of colon cancer and the anti-tumor immunity function,and shows an important clinical significance in predicting lymphatic metastasis of colon cancer. MSI and LOH may separately control the development of sporadic colon cancer with different pathways. LOH mostly arises in the late period of sporadic colon cancer and endows a high aggressive and poor prognostic phenotype. By compassion, MSI may be an early period molecule marker for sporadic colon cancer, enhanced expression of nm23H1 protein can effectively inhibit colon cancer metastasis and improve prognosis of sporadic colon cancer patients.     

燃煤型砷中毒患者皮损PTCH基因D9S287和D9S180位点微卫星不稳定性的观察

目的观察燃煤型砷中毒患者皮损组织中PTCH基因微卫星DNA不稳定性及杂合性丢失与临床病理、临床分度之间的关系。方法选取D9S287、D9S180两个微卫星多态性标记,采用PCR扩增-变性聚丙烯酰胺凝胶电泳-银染法检测不同病理类型的燃煤型砷中毒患者的微卫星的改变。结果34例患者皮损组织PTCH基因微卫星不稳定性的发生率为29.41%(10/34),杂合性丢失的发生率为14.7%(5/34),微卫星的改变与病理分型相关(P<0.01),与临床分度无关(P>0.05)。结论PTCH基因微卫星不稳定性和杂合性丢失可能在砷中毒患者皮损癌变的发生发展中起重要作用。     

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.     

Microsatellite analysis of induced sputum DNA in patients with lung cancer in heavy smokers and in healthy subjects

Abnormality in the fragile histidine triade (FHIT), a candidate tumor suppressor gene located in chromosome region 3 (3p14.2), has been frequently found in multiple tumor types, including lung cancer. In this study, the authors assessed the consistency of DNA microsatellite analysis of induced sputum (IS), as compared to that of blood and plasma. They also evaluated the loss of heterozigosity (LOH) and microsatellite instability (MSI) in 3 different loci, D3S1300, D3S1313, and D3S1234, all internal to the FHIT gene, in IS, blood, and plasma from patients with lung cancer, smokers, and healthy subjects. Eighteen patients with lung cancer (3 females, age mean +/- SD: 63 +/- 7 years), 39 smokers (23 females, age mean +/- SD: 57 +/- 6 years and cigarette pack-years mean +/- SD: 34 +/- 12), and 22 healthy nonsmoking subjects (13 females, age mean +/- SD: 63 +/- 5 years) were studied. DNA was extracted from blood, plasma, and IS, by means of a standard method. Analysis of LOH and MSI were performed using a fluorescent polymerase chain reaction (PCR)-based approach, followed by capillary electrophoresis. The ratios between the peak heights (phs), expressed as random fluorescence units, from plasma/blood (p/b) and induced sputum/blood (is/b) in all three loci were considered. The biases (agreement limits) between the mean ph ratio from p/b and is/b of D3S1300, D3S1313, and D3S1234 were respectively 0.07 (- 0.39 to 0.53), 0.016 (- 0.32 to 0.35), - 0.10 (- 0.51 to 0.30) in the patients; - 0.04 (- 0.52 to 0.43), - 0.06 (- 0.31 to 0.18), - 0.08 (- 0.48 to 0.30) in smokers; and - 0.11 (- 0.40 to 0.17), - 0.05 (- 0.53 to 0.43), - 0.09 (- 0.51 to 0.33) in healthy subjects. LOH and MSI in at least one locus were observed in 55% of patients, in 18% of smokers, and in 4.5% of healthy subjects (P < 0.001). These results showed that IS DNA provided data that were consistent with those from blood and plasma. These findings highlight new prospects for early tumor detection by a noninvasive technique based on the analysis of genetic alterations in induced sputum.