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Cell proliferation and viability were assayed 48h after transfection, and MDA-7 demonstrated selective inhibition of tumor cell growth, inhibitory rates of A549, Hela and HepG2 cell lines were 25%, 20% and 19%, respectively, but had no significant effect on human fetal kidney derived 293 cell line. Hela cell was screened by G418 for 2 weeks after pcDNA3-MDA-7 and monolayer colony was counted, its monolayer colony formation was 30% of cells transfected with pcDNA3. 0 vector. CMV-driven MDA-7 adenovirus vector was constructed. 293 showed no significant apoptosis during adenovirus packaging and the unpurified adenovirus titer was about 1×10〓pfu/ml. Cos 7, A549, Hela, HepG2 and Hep3B cell was infected with Ad-GFP at different MOI.

二。黑色素细胞分化相关蛋白-7（MDA-7）的克隆及功能研究：利用RT-PCR方法从5月龄人胚胎脾细胞扩增MDA-7的编码序列，经测序鉴定序列与文献报道一致后，与真核表达载体pcDNA3.0连接，构建pcDNA3-MDA-7表达载体，瞬时转染293、A549、Hela和HepG2细胞后抽提细胞总RNA，RT-PCR结果显示表达载体可介导MDA-7在不同细胞系中有效表达；转染48h后测定细胞增殖和活力，MDA-7可选择性抑制肿瘤细胞的增殖，对A549、Hela和HepG2细胞的抑制率分别为25％、20％和19％，但是对人胚肾来源的293细胞生长无明显影响。pcDNA3-MDA-7载体转染Hela细胞后，以G418筛选2周后计数单层细胞集落形成数，计数仅为转染pcDNA3.0空载体的细胞的30％左右。

The ELISA titre was 1:2000.By cell fusion, 46 hybridoma cell lines were screened,and 10 lines were cloned with limited dilution method.16 lines secreting anti-bFGF monoclonal antibody were been developed, and 2 lines targeted fusion protein. Sensitive ELISA and dot-ELISA for bFGF was developed with this mAb. The detection limit of them were 0.1 ng/well and 0.5 ng/well. The expression level of anti-bFGF mAb by different rebuilt engineering cells were identified by western blot and to direct rebuild recombiment engineering cell. The dose and character of anti-bFGF mAb inhibiting bFGF biology activity were searched by 3T3 cell line. Searching 20 tissue of liver cancer, liver cancer cell lines and general tissue of liver, finding bFGF were highly expressed in tissues of liver cancer and liver cancer cell lines. Affinity chromatography purifying bFGF was set up by mAb binging CNBr-pepharose 4B, and the purification was 95%. We found that the titer of anti-bFGF antibody was very high in serum of neuropathic amyotrophia.

应用细胞融合制备46株杂交瘤，对其中10株进行克隆化，获得bFGF特异单抗16株，2株针对融合蛋白；应用该单抗建立了0.1ng/孔灵敏度的ELISA，0.5ng/孔敏度的斑点ELISA；用Western-blotting鉴别了经改造不同工程菌蛋白表达，指导重组工程菌改造；用3T3细胞培养研究了单抗抑制bFGF生物学活性的剂量和特点；合作研究了20例肝癌、肝癌细胞株和正常肝组织，发现前者bFGF高表达；应用单抗偶联CNBr-sepharose 4B建立了小量免疫亲和层析纯化bFGF，纯度达到95%；发现神经性肌萎缩患者血清中含有高滴度的bFGF抗体，已有10多家单位引用单抗或进行合作。

Pathology findings:there were 294 cases of clear renal cell carcinoma (83.76%),including 2 cases of cystis renal cell carcinoma ,27 cases of Grannular renal cell carcinoma ( 7.69 %), 14 cases of papilliform renal cell carcinoma , 6 cases of the mixed type renal cell carcinoma,10 cases of other cell types .

病理结果：透明细胞癌294 例(83.76%，其中囊性肾细胞癌2例)，颗粒细胞癌27 例(7.69%)，乳头状肾细胞癌14例(3.98%)，肾混合细胞癌6例(1.7%)，其它癌10例。

Pathology findings:there were 294 cases of clear renal cell carcinoma (83.76%),including 2 cases of cystis renal cell carcinoma ,27 cases of Grannular renal cell carcinoma ( 7.69 %), 14 cases of papilliform renal cell carcinoma , 6 cases of the mixed type renal cell carcinoma,10 cases of other cell types . Followup was achieved in 263 cases (74.93％),with tie 3、5year and 10 year survival rates being 70.45 %(124/ 176),53.3%(40/75) and 23.8%(5/21) respectively.

病理结果：透明细胞癌294 例(83.76%，其中囊性肾细胞癌2例)，颗粒细胞癌27 例(7.69%)，乳头状肾细胞癌14例(3.98%)，肾混合细胞癌6例(1.7%)，其它癌10例。263 例(74.93 %)获得随访，随访时间6个月～12 年，3 年、5 年和10年生存率分别为70。

Pathology findings:there were, 294 cases of clear renal cell carcinoma （83.76 %）, including 2 cases of cystis renal cell carcinoma , 27 cases of Granntdar renal cell carcinoma （ 7.69 %）, 14 cases of papilliform renal cell carcinoma , 6 cases of the mixed type renal cell carcinoma, 10 cases of other cell types .

病理结果：透明细胞癌294例（83.76%，其中囊性肾细胞癌2例），颗粒细胞癌27例（7.69%），乳头状肾细胞癌14例（3.98%），肾混合细胞癌6例（1.7%），其它癌10例。263例（74.93%）获得随访，随访时间6个月~12年，3年、5年和10年生存率分别为70.45%（124/176）、53.3%（40/75）和23.8%（5/21）。

The results demonstrated that, by comparison with un-transgenic Roselle cell or callus, the critical plating cell density and critical initiating cell density of transgenic cell line were cut down 60% and 50%, respectively,the growth cycle of transgenic cell in suspension culture was shortened from 16 days to 12 days, and the specific growth rate of transgenic Roselle callus was raised 75%, the content of flavonoid compounds in transgenic Roselle cell line or callus was hardly altered.

研究结果表明，所获得的转基因玫瑰茄细胞系的临界植板细胞密度和临界起始细胞密度分别降低了60%和50%，悬浮细胞培养周期由16d缩短到12d，转化愈伤组织的比生长速率比对照提高了75%，转基因玫瑰茄细胞和愈伤组织中的花黄素含量与对照相比没有发生明显变化。

The efficiency of selection was monitored by comparing the number of phage recovered from the acid elution and cell lysate in each round,and by testing EGFR binding specificity of polyclonal phagescFv on CHOEGFRGFP1 and CHOK1 cell with cell ELISA. Bacterial PCR was used to select clones containing a 1 kb insert. Cell ELISA was used to determine EGFR binding specificity of monoclonal pscFv on EGFR positive and negative cell. The number of individual EGFRbinding clones was determined with nucleotide sequencing. Results 500fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection.

以稳定转染的CHOEGFRGFP1细胞和未转染的CHOK1细胞分别作为EGFR阳性和阴性细胞，采用负筛选的方法进行筛选；通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆pscFv与阳性、阴性细胞结合情况对筛选过程进行监测；采用菌落PCR挑选含有全长scFv片断的菌落，进一步用细胞ELISA检测单克隆pscFv与EGFR阳性及阴性细胞结合特异性；挑选EGFR特异性单克隆pscFv采用DNA测序法确定克隆多样性。

More materil as took from resected specimen is helpful for finding more small cell carcinoma,there is poor prognosis in small cell carcinoma concomitant with squamous cell carcinoma compared with squamous cell carcinoma alone,it is suggested that primary small cell carcinoma of esophagus might be derived from a totipotentiality primitive cell in the basal region of the squamous mucosa of the esophagus.

对手术切除标本足够取材有利于发现肿瘤的异源性成分；小细胞癌合并鳞癌者，预后较单纯鳞癌者差；本文结果支持食管小细胞癌起源于原始多潜能干细胞的观点。

Result and contrast group quite, active of CD4+T/CD8+T value and cell of the CD4+T before observing group method, NK cell is reduced apparently, and CD8+T cell is elevatory, after concerning; technique in installment with TNM 2 weeks relatively active of CD4+T/CD8+T value and cell of the CD4+T before art, NK cell is apparently elevatory, and the 4 A after CD8+T cell reduces; art is followed visit discovery, after afore-mentioned index change trends are the same as art 2 weeks, lift without recrudescent transferrer or reduce scope more remarkable.

结果和对照组比较，观察组术前CD4+T细胞、CD4+T/CD8+T值及NK细胞活性明显降低，而CD8+T细胞升高，和TNM分期有关；术后2周较术前CD4+T细胞、CD4+T/CD8+T值及NK细胞活性明显升高，而CD8+T细胞降低；术后4 a随访发现，上述指标变化趋向同术后2周，无复发转移者升高或降低幅度更显著。

In Part 1 of our research, BGSCs were sorted through immunomagnetic beads marking by CD133 and cultured in vitro, and character as a stem cell was identified by stem cell markers (CD133 and Nestin) and differentiated cell markers [ microtubule-associated protein 2(MAP2), glial acidic fibrillary protein and myelin basic protein] , ultrastructure observing with electron microscopeand engrafting to severe combined immunodeficiency mice for tumorigenesis test. The results were as following: Only a small subset of CD133+ glioma cells in glioma cell lines and fresh specimens from various pathologic grade could express stem cell markers CD133 and Nestin, view ultrastructure of a stem cell and be capacity of serial passage in culture. These CD133+ cells possese a marked capacity for multipotent differentiation and could differentiate into tumor cells expressing MAP2,β-TubulinⅢ, GFAP and MBP; When engrafted into SCID mice, they can generate and form tumors that phenotypically resembl the tumor from the patient.

在本课题中，在第一部分实验中采用以CD133为标志的免疫磁珠法从人脑胶质瘤组织和细胞株中分离脑胶质瘤干细胞并进行体外培养，通过免疫荧光技术检测干细胞标志物CD133、Nestin，诱导分化后检查分化细胞标志物MAP2、GFAP、MBP以及电镜超微结构观察和移植SCID鼠致瘤实验，对其干细胞特性加以鉴定，得到如下结果：不同病理分级的新鲜胶质瘤标本和胶质瘤细胞株中存在一小部分CD133+的胶质瘤细胞，能表达干细胞的标志物CD133和Nestin，符合干细胞的超微结构特点，体外培养能连续传代；具有多向分化潜能：诱导分化后能产生MAP2、β-TubulinⅢ、GFAP、MBP染色阳性的细胞；移植SCID鼠后能形成与亲本肿瘤表型一致的移植瘤。

They have quite a lot of work to do.

他们有许多工作要做。

Let us give you a more elaborate example of this process.

让我们给你这个过程的更多的详细例子。