应用噬菌体随机肽库筛选结肠癌患者血清中的肿瘤标记物的研究

目的用噬菌体随机肽库技术对结肠癌病人血清进行差异性筛选,以获得特异性的肿瘤标志物。方法用噬菌体随机十二肽库与结肠癌病人血清和正常人血清进行淘选,经过几轮吸附和洗脱得到重组噬菌体库。采用ELISA方法进行单克隆鉴定,挑选20个阳性克隆测序获得相同的序列:19B(GSMSRYVRWYTP),人工合成小肽19B的寡核苷酸序列,并将其与原核表达载体PGEX-4T-I连接,转化大肠杆菌BL-21(DE3),诱导融合蛋白的表达,以Glutathione Sepharose 4B进行纯化。结果用纯化的蛋白包被抗原板检测24例正常人血清和24例结肠癌病人的血清,证实小肽在正常人血清和结肠癌病人血清间存在差异。结论小肽19B有可能作为检测结肠癌的生物标志物。

血清结肠癌转移相关基因-1在晚期胃癌组织中的表达及对阿帕替尼化疗疗效的预测价值

目的探究血清结肠癌转移相关基因-1(MACC1蛋白)在晚期胃癌组织中的表达及对阿帕替尼化疗疗效的预测价值。方法选取60例晚期胃癌患者,按治疗方法的不同将其分为对照组(n=36)和观察组(n=24)。对照组给予替吉奥片、奥沙利铂甘露醇注射液治疗,观察组在此基础上加用阿帕替尼片,所有患者均化疗3个周期。统计MACC1蛋白在正常胃黏膜组织和胃癌组织中的表达情况;统计化疗前和化疗3个周期后2组患者MACC1蛋白含量,统计2组患者化疗3个周期后的预后情况。结果MACC1蛋白在胃癌组织中的阳性表达率显著高于正常胃黏膜组织中的表达率;化疗3个周期后,2组患者的MACC1蛋白含量均降低,且观察组较对照组降低的更显著,观察组的无进展生存时间和总生存时间均显著长于对照组(P<0.01)。结论MACC1有助于胃癌的诊断,且MACC1越低阿帕替尼的化疗效果越好。

Detection of Serum Aberrant CDKN2/P16 DNA in Colorectal Cancer

Objective： To search for a biomarker for colorectal cancer. Methods： The MSP, SSCP and deletion tests with serum have been taken simultaneously in 100 cases of colorectal cancer and 2 groups of controls, as well as the specimens of 26 cancer tissues and 22 paracancerous tissues and 29 cases of benign disease tissues for a contrast. Results： The aberrant methylation rate of P16 in the serum was 69.00%, deletion rate 4.00% and suspicious point mutation rate 15.00% in colorectal cancer patients. The data of cancer tissues were the same as those of the serum, but in paracancerous tissue those were significantly lower. In 10 cases, sequencing analysis revealed that there were 3 cases of missense, one case of frameshift and one case of nonsense. Among them, four cases had P16 protein deletion. As a tumor marker, the sensitivity of combined use of three methods was 88.00%, specificity 96.87% and accuracy 90.15%. The combined use of MSP and SSCP could obtain the same results. Conclusion： The content of DNA in serum is minimal, but it reflects the tumor burden of patients. The 10^-3 fragments of DNA could be detected in the serum by MSP. It can be used in the clinical diagnosis or popular investigation, and long-term postoperative follow-up.

Detection of RASSF1A promoter hypermethylation in serum from gastric and colorectal adenocarcinoma patients

AIM：To evaluate the diagnostic role of serum RASSF1A promoter hypermethylation in gastric and colorectal adenocarcinoma. METHODS：Methylation-specific polymerase chain reaction （MSPCR） was used to examine the promoter methylation status of the serum RASSF1A gene in 47 gastric adenocarcinoma patients, 45 colorectal adenocarcinoma patients, 60 patients with benign gastrointestinal disease （30 with benign gastric disease and 30 with benign colorectal disease）, and 30 healthy donor controls. Apaired study of RASSF1A promoter methylation status in primary tumor, adjacent normal tissue, and postopertive serum were conducted in 25 gastric and colorectal adenocarcinoma patients who later were underwent surgical therapy. RESULTS：The frequencies of detection of serum RASSF1A promoter hypermethylation in gastric （34.0%） and colorectal （28.9%） adenocarcinoma patients were significantly higher than those in patients with benign gastric （3.3%） or colorectal （6.7%） disease or in healthy donors （0%） （P 〈 0.01）. The methylation status of RASSF1A promoter in serum samples was consistent with that in paired primary tumors, and the MSPCR results for RASSF1A promoter methylation status in paired preoperative samples were consistent with those in postoperative serum samples. The serum RASSF1A promoter hypermethylation did not correlate with patient sex, age, tumor differentiation grade, surgical therapy, or serum carcinoembryonic antigen level. Although the serum RASSF1A promoter hypermethylation frequency tended to be higher in patients with distant metastases, there was no correlation between methylation status and metastasis. CONCLUSION：Aberrant CpG island methylation within the promoter region of RASSF1A is a promising biomarker for gastric and colorectal cancer.

Proteome of human colon cancer stem cells:A comparative analysis

AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.

Identification of serum proteins discriminating colorectal cancer patients and healthy controls using surface-enhanced laser desorption ionisation-time of flight mass spectrometry

AIM： To detect the new serum biomarkers for colorectal cancer （CRC） by serum protein profiling with surfaceenhanced laser desorption ionisation - time of flight mass spectrometry （SELDI-TOF MS）. METHODS： Two independent serum sample sets were analysed separately with the ProteinChip technology （set A： 40 CRC ＋ 49 healthy controls; set B： 37 CRC ＋ 31 healthy controls）, using chips with a weak cation exchange moiety and buffer pH 5. Discriminative power of differentially expressed proteins was assessed with a classification tree algorithm. Sensitivities and specificities of the generated classification trees were obtained by blindly applying data from set A to the generated trees from set B and vice versa. CRC serum protein profiles were also compared with those from breast, ovarian, prostate, and non-small cell lung cancer. RESULTS： Mass-to-charge ratios （m/z） 3.1×10^3, 3.3× 10^3, 4.5×10^3, 6.6×10^3 and 28×10^3 were used as classitiers in the best-performing classification trees. Tree sensitivities and specificities were between 65% and 90%.Host of these discriminative m/z values were also different in the other tumour types investigated. M/z 3.3× 10^3, main classifier in most trees, was a doubly charged form of the 6.6× 10^3-Da protein. The latter was identified as apolipoprotein C-I. M/z 3.1×10^3 was identified as an N-terminal fragment of albumin, and m/z 28× 10^3 as apolipoprotein A-I. CONCLUSION： SELDI-TOF MS followed by classification tree pattern analysis is a suitable technique for finding new serum markers for CRC. Biomarkers can be identified and reproducibly detected in independent sample sets with high sensitivities and specificities. Although not specific for CRC, these biomarkers have a potential role in disease and treatment monitoring.

miR-93 suppresses proliferation and colony formation of human colon cancer stem cells

AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum- free medium. A miRNA microarray was used to detect differential expression profiles of rniRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction （PCR） was performed to verify the dif- ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic- tion tools, miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription （RT）-PCR and western blotting.RESULTS： Compared with expression in SW1116 cells, 35 miRNAs （including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa- miR-32） were upregulated more than 1.5-fold, and 11 miRNAs （including hsa-miR-93, hsa-miR-1231, hsa- miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561） were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKNIA, HDACS, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHCl. Overexpressed miR-93 sig- nificantly inhibited cell proliferation and colony forma- tion by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION： Some miRNAs were differentially ex- pressed during differentiation of SW1116csc into SW1116 cells, miR-93 may inhibit SW1116csc proliferation and colony formation.

Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas

AIM: Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors,and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals.METHODS: The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC.RESULTS: The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P＜0.05).The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract.CONCLUSION: These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.