Objective: To establish a fluoregenic probe quantitative RT-PCR (FQ-RT-PCR) method for detection of the expression of MDR1 gene in tumor cells and to investigate the expression of MDR1 gene in patients with lung cancer. Methods: The fluorogenic quantitative RT-PCR method for detection of the expression of MDR1 gene was established. K562/ADM and K562 cell lines or 45 tumor tissues from patients with lung cancer were examined on PE Applied Biosystems 7700 Sequence Detection machine. Results: the average levels of MDR1 gene expression in K562/ADM cells and K562 cells were (6.86±0.65)× 107copies/mg RNA and (8.49±0.67)×105 copies/mg RNA, respectively. The former was 80.8 times greater than the latter. Each sample was measured 10 times and the coefficient variation (CV) was 9.5% and 7.9%, respectively. Various levels of MDR1 gene expression were detected in 12 of 45 patients with lung cancer. Conclusion: Quantitative detection of MDR1 gene expression in tumor cells was achieved by using FQ-RT-PCR. FQ-RT-PCR is an accurate, and sensitive method and easy to perform. Using this method, low levels of MDR1 gene expression could be detected in 24% of the patients with lung cancer.

作者：高劲松；马刚；仝明；陈佩毅；王传华；何蕴韶 刊期： 2001年第02期

Objective: To elucidate the expression and significance of cell cycle protein cyclin D1 in human hepatocellular carcinoma (HCC). Methods: The expression of cyclin D1 protein in 29 cases of HCC tissues was detected by immunohistochemical ABC method, and the relationship between its positive rate and pathological grades of HCC tissues was analyzed. Results: The positive rate of cyclin D1 in HCC tissues was 58.6%(17 in 29 cases), whereas only 18.2% (2 cases of 11 cases) in the non-tumor liver tissues immediately adjacent to HCC tissues (LAH). There was significant difference between grade II and LAH tissues (P<0.05), and between grade I and grade III on the positive rate of cyclin D1 (P<0.05), respectively. Conclusion: Cyclin D1 may be regarded as an oncogenic marker during the genesis and development of HCC, and its role in the transforming process from G1 phase to S phase of HCC cells needs further studies.

作者：杨连君；司晓辉 刊期： 2001年第02期

Objective: To screen LKB1 mutation in sporadic colon and ovarian tumors. Methods: Using PCR-SSCP analysis, 72 colon cancer, 45 ovarian cancer, 14 granulosa cell tumor were screened for LKB1 mutation. Results: no mutation was in sporadic colon and ovarian adenocarcinomas. Two mutations were detected in one of the granulosa cell tumors: a mis-sense mutation affecting the putative start codon (ATG?ACG, MIT); and a silent change in erxon 7 (CTT?CTA, leucine). Conclusion: LKB1 mutations in sporadic colon and ovarian cancers are rare event and LKB1 is not the target gene lost on chromosome 19p13.3 in ovarian cancers.

作者：王振军；严仲瑜；万远廉；郭彦彦；徐文怀；XU Wen-huai 刊期： 2001年第02期

Objective: To clone multidrug resistance (MDR) related genes in lung adenocarcinoma cell lines. Methods: The differentially expressed cDNA fragments between A549 and A549DDP cells were analyzed by mRNA differential display PCR(DD RT-PCR). The fragments thus obtained were further analyzed by DNA sequencing and Northern blotting. Results: Three differentially expressed cDNA fragments were obtained and confirmed by Northern blot. Sequence analysis revealed that two of them were novel and one was 100% identical with ICE gene. Conclusion: Analyzing differentially expressed fragment between A549 and A549DDP cells may be helpful for finding new MDR related genes. The drug resistance of A549DDP cells may be related to the inhibition or down-regulation of ICE gene.

作者：王洁；刘叙仪；李西平；李振甫；张宏 刊期： 2001年第02期

Objective: Evaluation the role of neoadjuvant chemotherapy in invasive thymoma. Methods: 14 patients with invasive thymoma(Masaoka stage Ⅲ in 12 and Ⅳa in 2) were treated with neoadjuvant chemotherapy by CAVP for 3-4 cycles (cyclophosphamide 600mg/m2 D1, adramycin 30mg/m2 or epi-adramycin 40mg/m2 D1, vincristine 0.6mg/m2 D1 or vindestine 3mg/m2 D1、D8, cisplatin 30mg/m2 D1、2、3)。After chemotherapy, all patients underwent operation in 1-3 months. We performed 10 sternotomies and 4 anterolateral thoracotomies. Radiotherapy was administrated with total dose 50-60Gy in all patients except for those who were pathologically complete remission. The group was followed up by 6 months to 3 years. Results :After chemotherapy, 5 patients(35.7%) had a complete remission(CR) and 9 patients(64.3%) had a partial remission(PR)。At operation, 9 patients had radical resection and 5 had partial resection. Postoperative pathological examination found only fibrosis in 5 patients with CR by chemotherapy. All patients were still alive except 2 patients died from visceral metastasis after 18 months and 2 years respectively. Conclusion: Neoadjuvant chemotherapy can increase the resectability of stage Ⅲ and Ⅳa invasive thymoma. A longer follow-up and a larger number of patients are needed to determine the impact of this treatment on long-term survival.

作者：谭黎杰；仇德惠；王群；徐正浪；徐松涛 刊期： 2001年第02期

Objective: To investigate the effects of anti-PML (promyelocytic leukemia) or anti-PML/RARa (promyelocytic leukemia/retionic acid receptora) antisense oligonucleotides on cell growth, expression of PML-RARa mRNA and PML-RARa/PML protein location of NB4 cell lines. Methods: RT-PCR was used for detecting PML-RARa mRNA expression, trypan blue exclusion for cell count, methylcellose assay for leukemic colony forming unit detection, immuno- fluorescence for PML-RARa/PML protein location. Results: Both anti-PML start codon region antisence (STAS) and anti-PML-RARa fusion region antisence (FUAS) could inhibit cell growth and the formation of acute myelocytic colony forming unit of cells(AML-CFU). Cells become partial differentiated at days 5, being more obvious in FUAS-treated cells than in STAS ones. Down regulation of PML-RARa mRNA expression occurred at 24 hours in STAS and FUAS-treated cells and maintained for up to 72 hours. Immuno-fluorescence analysis with anti-PML monoclonal antibody showed a remarkable decrease even complete disappearance of microgranules. The residual granules became enlarged as discrete dots (<10 per cell), similar to normal POD structure in some STAS-treated cells at 24 hours. At 72 hours, nearly all the granules disappeared. Similar changes were observed in FUAS-treated cells. Conclusion: Both PML and PML-RARa antisence oligonucleotides can specially block the expression of PML-RARa at mRNA and protein levels. PML protein is implicated in the regulations of cell differentiation.

作者：陈烨；缪金明；方智雯；朱学宏；邵念贤；欧阳仁荣 刊期： 2001年第02期

Objective: To screen novel genes related to adriamycin (Adr) resistance from human ovarian cancer resistance cell line OC3/Adr. Methods: Multidrug resistant ovarian cancer cell line OC3/Adr was induced by intermittent treatment of the human parent cell line OC3 with high concentration Adr. The difference of gene expression was screened by using different display analysis to the acquired Adr-resistance subline OC3/Adr and its parent cell line OC3. Results: OC3/Adr cell line was obtained which was more resistance to Adr than the parent cell line OC3 with the resistance index (RI) of 15.4. The OC3/Adr cell line also showed cross-resistance to other anti-cancer drugs (VP16, CDDP,5FU ). It grew slowly and exhibited changes of cell cycle. A number of differentially expressed ESTs (Expressed Sequence Tags, ESTs) were identified at mRNA level between the OC3/Adr and OC3. Four of 18 different ESTs were sequenced. The 431/432 base pair S1 was homologous to human sperm zona pellucida binding protein, while the other two ESTs, S3 and S4, were new gene segments, which were registered to GenBank with the number of AF 117656 and AF 126507 respectively. Particularly, the expression of S2 sequence increased in all the drug-resistance cell lines and S3 sequence overexpressed in human ovarian cancer tissues as compared with benign ovarian tumors. Adr in ovarian cancer OC3/Adr is involved with changes of multiple gene expressions.

作者：田方；程国均；周海胜；王宏；肖凤君 刊期： 2001年第02期

Objective: To understand whether verapamil (VER) resistance development in the multidrug-resistant cell line and its mechanism. Methods: K562/ADM/VER cell subline resistant to verapamil was established through a gradual increase of VER concentration in the media. MTT method was used to assay resistance to VER, cross resistance to dipyriamole (DPM), cyclosporin A (CsA) in the cells, and HPLC and spectrofluorometer to detect intracellular accumulation of VER or ADM respectively, as well as S-P immunocytochemical technique for detection of genes expression. Results: It were observed that 7.9-fold increase in VER resistance, significantly reduced intracellular accumulation of VER or ADM and also develop across resistance to DPM and CsA in K562/ADM/VER cells, compared with its parent cell, K562/ADM. High-level of p-glycoprotein(pgp), middle-level of p53, p16, was present in two cell lines without expression of GSTPI, C-myc, C-myc, C-fos and C-erbB-2. Bc1-2 protein expression was found only in K562/ADM cells. Conclusion: K562/ADM cells were capable of being induced to develop resistance to VER.

作者：谢佐福；周冬梅；林贤东；林声；吴允昆 刊期： 2001年第02期

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

作者：李金华；梁念慈；莫丽儿；张晓；何承伟 刊期： 2001年第02期

Objective: To detect the effect of arsenic trioxide or ATRA on APL cells or HL-60 cells and to investigate the mechanism of the hyperleukocytosis and detect the cross resistance between ATRA and arsenic trioxide. Methods: The number of promyelocytes or more matured granulocytes were counted by regular method, MTT test was used to measure the proliferation of HL-60 cells or APL cells, flow cytometry analysis to measure the apoptosis, NBT method to detect the differentiation of HL-60 cells or APL cells. Results: The proliferation of primary APL cells or HL-60 cells could be inhibited in vitro by either arsenic trioxide or ATRA, which could induce obvious apoptosis or obvious differentiation of primary APL cells or HL-60 cells. Inhibition of proliferation or apoptosis of ATRA resistant HL-60 cells were achieved by exposure toarsenic trioxide in vitro. On the other hand, the results of in vivo treatment showed that arsenic trioxide also induce of hyperleukocytosis. Conclusion: The results indicated that the hyperleukocytosis induced by ATRA is not contributed to the mechanism of more differentiation than apoptosis, there was not cross resistance between ATRA and arsenic trioxide.

作者：姜国胜；唐天华；孙关林；邬维礼；赵良玉；任海泉；董强；任青华；姜枫勤；JIANG Feng-qin 刊期： 2001年第02期

Objective: To introduce the method of a modified transcranial approach for resection of paranasal sinuses tumors involving the anterior skull base and to address our experience with the approach. Patients and Methods: Ten cases were operated by the approach. Among them, 4 suffered from benign meningeomas, 6 with malignant tumors included one chondrosarcoma, two malignant meningeomas, two olfactory neuroblastomas, and one squamous sarcoma. Of the patients, 4 cases had primary tumor and 6 cases had recurrent tumors. Result: All of the ten cases underwent operation and no postopertion complication occurred. 7 cases have survived for one to four years without tumor recurrence. 3 cases with malignant tumor died of tumor relapse in one to two years. Conclusion: This method significantly has helped to reduce the persistence and recurrence of the disease.

作者：赵素萍；陶正德；肖健云 刊期： 2001年第02期

Objective: To investigate the relationship between the prognosis of Epithelial Ovarian Cancer (EOC) and its ascites. Methods: Retrospectively analysis is performed for the clinical, pathological and followed up data of 101 in-patients suffering from epithelial ovarian cancer and operated with tumor debulking surgery in our hospital from January 1986 to December 1993. The patients was divided into two groups based upon the first laparotomy finding with ascites(62) or without(39). Age average, cell type, advanced proportion and survival rate of the patients are evaluated by a c2 test. Results: For age average and cell type, no statistical difference was noted. However, there were more advanced cases in ascites group than in the other (P<0.01). The 3-, 4- and 5-year survival in the no-ascites group were 87.02%, 73.42%, 57.10% respectively compared with 65.02%, 38.66%, 28.12% in the ascites group. The 5-year survival rate of stage I, II,III, IV patients in no-ascites group are 77%, 70%, 41.1%, 0 respectively, compared with that of 60%, 56.8%, 15.46%, 0 respectively in the ascites group. The results shows that 3-, 4-, and 5-year survival in no-ascites group were significantly higher than those in ascites group(P<0.01). Conclusion: Presence of ascites is a factor of poor prognosis for EOC.

作者：宋水勤；张国楠；吴艳丽；周红；赵素兰；谢方；陈毅男 刊期： 2001年第02期

Objective: To analyze the expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and its relation to angiogenesis. Methods: Tissue sections from 71 HCC patients were examined immunohistochemically for protein expression of iNOS, eNOS, and VEGF. Microvessal density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. Results: Positive immunostaining for iNOS, eNOS was detected in 83.1% and 85.9% of HCC respectively. INOS and eNOS were not detected in normal hepatic tissue. MVD was 34.3±1.5/HP and 38.6±1.6/HP in HCC with positive staining for iNOS and VEGF while it was 31.2± 2.8/HP, and 22.4± 2.0/HP in HCC with negative staining for iNOS and VEGF (P<0.01). A correlation between NOS expression and VEGF in HCC was not observed. Conclusion: iNOS and eNOS may play a role in malignant transformation f post-hepatic cirrhosis. The expression of iNOS and VEGF favors angiogenesis of HCC.

作者：王鲁；汤钊猷；孙惠川；叶胜龙；纪元；陆洪芬；施达仁 刊期： 2001年第02期

Objective: To eveluate major liver resections with vascular exclusion (TVE) in patients with hepatocellular carcinoma (HCC). Methods: Sixteen consecutive, major liver resections performed with TVE in HCC patients were analyzed retrospectively. The patients' mean ages were 54 years. Ten patients had cirrhosis and eleven patients had chronic hepatitis B. Results: There was no perioperative death and the mean hospital stay was 20 days and the average amount of intraoperative blood transfusion was 400 mL (range 0-2000 mL). Forty-four percent of the patients did not receive intraoperative blood transfusion. The mean total bilirubin(T-BIL) and aspartate aminotransferase (AST) were 24μmol/L (range 8-56μmol/L) and 56 IU/L (range 10-204 IU/L) measured prior to discharge. Conclusion: In our experience, total vascular exclusion is invaluable in major or difficult liver resections, especially when lesions adjacent to the hepatic veins and vena cava. It is associated with a low blood transfusion requirement and a low incidence of complications. It further obviates the need for dissection of the porta hepatis thus reduces the associated risks. Total vascular exclusion time of 30min appears to be well tolerated, even in patients with cirrhosis..

作者：季加孚；顾晋；苏向前；焦春雨；王怡；欧阳晓辉；董培德；杨成旺 刊期： 2001年第02期

Objective: To analyze cyclin D1 and cyclin- dependent kinase 4 (CDK4) expression and their significance in osteosarcoma of the jaws. Methods: Immunohistochemical ABC method was used to detect the expression of cyclin D1 and CDK4 in 20 cases of osteosarcoma and 8 cases of osteochondroma of the jaws. Results: The positive rates of cyclin D1 and CDK4 were 65% (13/20) and 60% (12/20), respectively. There was significant positive correlation between cyclin D1 and CDK4 expression (gs=0.48, P<0.05). Both cyclin D1 and CDK4 were present in 1/8 (12.5%) osteochondroma. The positive rate was remarkably different between osteosarcoma and osteochondroma (P<0.05). Conclusion: Cyclin D1 and CDK4 are overexpressed in osteosarcoma of the jaws and closely related to its occurrence and development.

作者：司晓辉；刘正 刊期： 2001年第02期

Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF1/CIP1 induction were detected by Western blot analysis. Northern blot analysis was carried out to examine whether changes in p21WAF1/CIP1 protein levels in MCF7 cells treated with AdCMVp53 are reflected at the mRNA level. Flow cytometric analysis of MCF7 cells following overexpression of recombination. Results: The ratio of p53: p21WAF1/CIP1 was below 1 at the early stages of AdCMVp53 infection, but increased to 1.6 by day 3 and to 9.7 by day 5 post-infection. As expected, p21WAF1/CIP1 expression was not detectable in MDA-MB-468 cells despite the presence of high levels of mutant p53 protein. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. By Northern blot analyzing the p21WAF1/CIP1: GAPDH ratios at different time points against the ratio at time point 0, a maximum 3-fold induction of p21WAF1/CIP1 mRNA expression relative to untreated control was observed on day 1 post-infection. The flow cytometric analysis indicated that MCF7 cells infected with AdCMVp53 undergo G1 arrest at both time points studied, with G1/S ratios ranging from 5.54 at day 1 to 5.65 at day 7. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. Conclusion: This studydemonstrated that p53 could regulate p21WAF1/CIP1 gene expression at both the transcriptional and post-transcriptional levels in MCF7 cells. The latter mechanism may be involved in or be responsible for, the induction of cell cycle arrest by transcription-defective mutants of p53.

作者：季加孚；张霁；焦春雨；顾晋；谭立新；张平；李培详 刊期： 2001年第02期

Objective: To find an effective, sensitive, specific and noninvasive diagnostic method of breast cancer. Methods: 109 masses of 102 patients with breast lesions smaller than 2 cm in diameter were divided into three groups to undergo 99mTc-MIBI imaging and compared with the results of pathology examination. 20 cases without breast lesions were selected as control. Abnormal condensation of 99mTc-MIBI in the breast reaching 10% higher than that in the counterpart of the healthy breast was regarded as positive. Results: Of 32 breast cancers, positive imaging appeared in 25. Negative imaging were found in 31 of 38 benign breast lesions. Of 39 occult breast lesions, positive imaging appeared in 6 and 3 of them were breast cancer, 2 of 3 patients with slightly increased 99mTc-MIBI imaging threshold were breast cancer also. No positive imaging was found in the control group. The diagnostic accuracy, sensitivity, specificity, positive predictive value, negative predictive value of 99mTc-MIBI was 88.4%, 89.2%, 88.0%, 75.0% and 95.3%, respectively. Conclusion: 99mTc-MIBI imaging had higher sensitivity and accuracy in the diagnosis of breast cancer and differentiation between benign and malignant breast lesions. It could provide useful information for the diagnosis of clinically suspected breast cancer.

作者：任长才；金少津；邹强；朱汇庆；王红鹰；梁春立 刊期： 2001年第02期

Objective: This study was designed to clone candidate tumor suppressor genes down-expressed in Nasopharyngeal Carcinoma (NPC). Methods: Differentially expressed cDNA fragments (AF152605 and AF091517) were labeled by PCR, and Northern blot was used to confirmed transcript length of these genes. Skeleton muscle cDNA library was screened with PCR-labeled probe mixture. By sequencing the positive clones directly, three novel genes (Genbank accession number: AF179285, AF170307 and AF194971), with transcripts of 2.1 Kb, 1.1 Kb and 1.4 Kb respectively, were isolated successfully. Conclusions: Library screening using PCR-labeled probes mixture is an efficient method to get full-length cDNA from multi-cDNA fragment simultaneously and quickly.

作者：余鹰；朱诗国；张必成；周鸣；李桂源；沈守荣；张晓梅 刊期： 2001年第02期

Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced combination of the two kinds of vaccine may have potential application value in human cancer treatment.

作者：张清媛；李殿俊；王志华 刊期： 2001年第02期