内皮抑素在脑胶质瘤中的表达及意义

目的:探讨内皮抑素(Endostatin,ES)在人脑胶质瘤中的表达及其意义。方法:采用免疫组织化学方法,对45例脑胶质瘤和5例正常脑组织标本中ES表达进行定位和定性分析。结果:高级别胶质瘤(Ⅲ~Ⅳ胶质瘤)ES表达水平明显高于低级别胶质瘤(Ⅰ~Ⅱ胶质瘤)和正常脑组织,差异有显著性意义(P<0.01);低级别胶质瘤(Ⅰ~Ⅱ胶质瘤)和正常脑组织ES表达水平差异无显著性意义(P>0.05)。结论:ES可能在人脑胶质瘤细胞侵袭性生长及恶性发展中起重要作用,且有可能与胶质瘤的恶性度及预后有关。     

比较两种免疫缺陷动物人肝癌模型的肿瘤生物学特性

为探讨裸小鼠和裸大鼠(rnu/rnu)两种免疫缺陷动物人肝癌皮下移植与原位移植后肿瘤生长和转移等生物学特性的差异,将Huh-7细胞株接种至裸小鼠皮下成瘤后采用组织学完整的组织块移植于裸小鼠和裸大鼠皮下和肝脏,建立皮下和原位移植模型,观察所建立模型的皮下和原位成瘤率、移植瘤生长、侵袭和转移情况,同时进行了病理组织学、超微结构、细胞增殖周期和异倍体的观察.裸大鼠皮下移植瘤模型肿瘤的生长速度远远大于裸小鼠.原位移植瘤模型裸大鼠成瘤率高达95.0%,其肺转移率形成率为50.0%,均高于裸小鼠.此实验证明应用裸大鼠较裸小鼠更适用于建立肝癌的皮下和原位移植模型,为探讨肝癌转移的生物学机制和抗转移治疗提供了理想的动物模型.     

New method for detecting cellular transforming genes

Tumor induction in athymic nude mice can be used to detect dominant transforming genes in cellular DNA. Mouse NIH 3T3 cells freshly transfected with either cloned Moloney sarcoma proviral DNA or cellular DNA's derived from virally transformed cells induced tumors when injected into athymic nu/nu mice. Tumors were also induced by cells transfected with DNA from two tumor-derived and one chemically transformed human cell lines. The mouse tumors induced by human cell line DNA's contained human DNA sequences, and DNA derived from these tumors was capable of inducing both tumors and foci on subsequent transfection. Tumor induction in nude mice represents a useful new method for the detection and selection of cells transformed by cellular oncogenes.     

人基质金属蛋白酶2(MMP-2)类血红素结构域克隆及原核表达

提取人神经胶质瘤U87细胞总RNA,通过RT-PCR克隆到人基质金属蛋白酶2（MMP-2）类血红素结构域pex的cDNA序列-PEX,连接pMD18-T载体和PEX,测序分析,构建原核表达载体PET42a-PEX,转化大肠杆菌BL21（DE3）,异丙基-βD-硫代半乳糖苷（IPTG）诱导后显示目的蛋白有37%的表达量,为进一步研究其抗肿瘤血管生成鉴定基础。     

裸小鼠人结肠癌术后转移模型的建立

将人结肠腺癌细胞株HCT-116接种于裸小鼠右侧背部近腋部皮下,得到皮下移植瘤,肿瘤在体内适应后用28只裸小鼠采用组织块法建立皮下移植瘤模型。4周后随机挑选15只裸小鼠作为实验组切除肿瘤组织,建立裸小鼠人结肠癌术后转移模型,其余13只裸小鼠作为对照组,观察动物生存情况及肿瘤远处转移情况。对照组裸小鼠9周时出现恶病质,解剖检查肺转移率为23%(3/13);实验组裸小鼠术部无肿瘤复发,17周后肺转移率达到100%(15/15),淋巴结转移率100%(15/15),脾脏转移率33.3%(5/15)。本模型模拟了临床肿瘤根除术后发生远处转移的过程,可为研究结肠癌转移机制和术后抗转移治疗提供理想的动物模型。     

Experimental Researches on Carcinogenesis or Tumorigenicity of MDCK Canine Kidney Cell(CKC) Lines and Analysis of Their Chromosome Karyotypes

The chromosomal number variations & structural aberrations of the MDCK cell line, primary feline or canine kidney cell(FKC or CKC) and Hela cell line were investigated and their karyotypes of conventional chromosome bands were analyzed. The carcinogenesis or tumorigenicity testing of these cell lines in about 232 nude mice and for colony formation in soft agarose and for haemagglutination under different concentration of plant lectins of these cells were carried out. Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with tetrapioid YA strain of MDCK cell during 20 - 45 passages, with hypodiploid JB strain of MDCK cell on passage 25, with di-and hypoploid JC strain of MDCK cell during 2 - 15passages or with hypoploid M strain of MDCK cell during 9 - 27 passages was 28/58, 1/5, 4/18 and 0/31,respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high (mcs + n) and lowest (mcs)passages was not more than 5 % - 15 % and the structure aberrations was generally 0 - 3%. These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. The repeatedly frozen, thawed and split controls of tumorigenicity-positive cell lines(X strain of Hela, M strain of BHK-21, JA strain of Vero, YA strain of MDCK) have much lower tumorigenicity or are even non-carcinogenesis, and the repeatedly frozen, thawed and split controls of very low tumorigenicity cell lines (M or JC strain of MDCK) are certainly non-carcinogenic and never have increased tumorigenicity.It is thus evident that MDCK cell of M, JB or JC strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YA strain can not be approved as substrate for the preparation of comattenuated viral vaccines. In summary, all strains of MDCK cell line have tunorigenicity, at least have low tumorigencity, never have non-cancinogenic MDCK, but very low tumorigenicity MDCK cell strains can certainly be used for the approval production of canine viral vaccines if the DNA content in viral cell cultures was remarkably decreased through conventional means in manufacturing process. Therefore, the master cell stock and working cell bank of MDCK line used for vaccine manufacture were established in China, which are free of infectious agents, and described with respect to cytogenetic characteristics and tumorigenicity. Tests showed that there were correlations among cell line chromosome number variations, anchorage independence in soft agarose, haemagglutination under plant lectins, and tumor-forming ability in nude mice, thus all the in vitro tests are economic, simple and reliable means for monitoring the tumor-forming ability of MDCK line in nude mice.     

人基质蛋白酶2(MMP-2)血红素结合蛋白样结构域原核表达、复性及生物学活性验证

用RT-PCR从人神经胶质瘤U87细胞扩增人基质金属蛋白酶-2(MMP-2)血红素结合蛋白样结构域(PEX)基因,克隆到pMD18-T载体然后测序;将PEX定向克隆到PET-25b(+)中构建原核表达载体PET-PEX,转化大肠杆菌BL21(DE3),诱导表达产生表达量近40%的包涵体蛋白,表达蛋白分子量大小约28.0 KD,与预期大小相符.包涵体经优化洗涤,8 mol/L尿素变性,采用透析法进行复性.鸡胚绒毛尿囊膜(CAM)试验显示复性后的PEX蛋白能显著抑制鸡胚新生血管生成(P<0.05),并且其抑制作用表现出一定的剂量依赖性.     

Human endometrial adenocarcinoma transplanted into nude mice: growth regulation by estradiol

A model for studying the growth of primary tumors of human endometrium and its regulation by 17 beta-estradiol has been developed in which ovariectomized nude mice are used as recipients. The receptors for sex steroids are maintained during serial transplantation of the tumor in this system. Although the rate of growth of receptor-negative endometrial tumors transplanted into ovariectomized nude mice is unaffected by the sustained presence or absence of estradiol, the growth of receptor-positive tumors is significantly increased by estradiol. Receptor-positive tumors treated with estradiol produced elevated concentrations of progesterone receptor. That the progesterone receptor is functional in this tumor is evident from the induction of estradiol 17 beta-dehydrogenase activity upon progestin administration. These findings are consistent with receptor-mediated regulation of growth of endometrial carcinoma.     

双歧杆菌完整肽聚糖对实验性胃癌抑制作用的研究

为研究双歧杆菌完整肽聚糖(WPG)对实验性胃癌生长的抑制作用,从两歧双歧杆菌中提取完整肽聚糖,以人胃癌裸鼠移植瘤为动物模型,观察不同浓度WPG对移植瘤生长的抑制情况,绘制肿瘤生长曲线,HE染色观察WPG对移植瘤组织形态的影响。结果显示,WPG低、中、高剂量组移植瘤的生长速度、平均瘤重均明显低于阴性对照组,抑瘤率分别为62.50%、67.87%和87.50%,与阳性对照(5-氟脲嘧啶)组相比无显著性差异(P>0.05)。HE染色结果显示,WPG组移植瘤中普遍存在不同程度的坏死。结果表明,双歧杆菌WPG能抑制裸鼠体内胃癌的生长,为开发新型高效、低毒的抗肿瘤药物提供试验依据。     

Establishment of Master Cell Stock and Working Cell Bank of MDCK Lines and Selection and Evaluation of the Lines as Candidate Viral Substrates for Approval Production of Combinational Canine Attenuated-live Virus Vaccines

Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0(0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with di-and hyperploid YB strain of MDCK cell during 17 - 23 passages, with hyper- and hypoploid KA strain of MDCK cell during 6 - 8 passages, with hypoploid WB strain of MDCK cell on passage 6, with hyper-and hypopioid H strain of MDCK cell during 8 - 24 passages was 2/24, 6/10, 5/10 and 10/15, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs + n) and lowest (mcs)passages was not more than 5- 15% and the structure aberrations was generally 0-3%. These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome karyotype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. It is thus evident that MDCK cell of WB or H strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YB or KA strain can not be approved as substrate for the preparation of attenuated viral vaccines.     

Tumorigenicity in human melanoma cell lines controlled by introduction of human chromosome 6

Chromosome banding analysis of human malignant melanoma has documented the nonrandom alteration of chromosome 6. To determine the relevance of chromosome 6 abnormalities in melanoma, a normal chromosome 6 was directly introduced into melanoma cell lines. The resulting (+6) microcell hybrids were significantly altered in their phenotypic properties in culture and lost their ability to form tumors in nude mice. The loss of the chromosome 6 from melanoma microcell hybrids resulted in the reversion to tumorigenicity of these cells in mice. The introduction of the selectable marker (psv2neo) alone into melanoma cell lines had no effect on tumorigenicity. These results support the idea that one or more genes on chromosome 6 may control the malignant expression of human melanoma.     

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.     

Pathogenicity of Klebsiella pneumonia(KpC4) infecting maize and mice

Recently, a new bacterial top rot disease of maize has frequently appeared in many areas of Yunnan Province, China. The pathogen of the disease was identified as Klebsiella pneumoniae(Kp C4), which is well known to cause pulmonary and urinary diseases in humans and animals and occasionally exists as a harmless endophyte in plants. To evaluate the virulence of the maize pathogen to maize and mice, we inoculated maize and mice with routine inoculation and intraperitoneal injection respectively according to Koch's postulates. The results showed that Kp C4 and the clinical strain K. pneumoniae 138(Kp138) were all highly pathogenic to maize and mice and the strain re-isolated from diseased mice also caused typical top rot symptoms on maize by artificial inoculation. It is highlighting that a seemingly dedicated human/animal pathogen could cause plant disease. This is the first report of K. pneumoniae, an opportunistic pathogen of human/animal, could infect maize and mice. The findings serve as an alert to plant, medical and veterinarian scientists regarding a potentially dangerous bacterial pathogen infecting both plants and animals/humans. The maize plants in the field could serve as a reservoir for K. pneumoniae which might infect animals and probably humans when conditions are favorable. The new findings not only are significant in the developing control strategy for the new disease in Yunnan, but also serve as a starting point for further studies on the mechanism of pathogenesis and epidemiology of K. pneumoniae.     

Angiogenic properties of Kaposi's sarcoma-derived cells after long-term culture in vitro

Cells derived from lung biopsies and pleural effusions from AIDS patients with Kaposi's sarcoma (KS) of the lungs were established in long-term culture with the aid of conditioned medium from HTLV-II-transformed T cells (HTLV-II CM). These AIDS-KS cells were similar to the so-called spindle cells in KS lesions and had some of their features. They produced factors that supported their own growth (autocrine) and the growth of other cells (paracrine), including umbilical vein endothelium and fibroblasts. That the AIDS-KS cells also expressed potent angiogenic activity was demonstrated by the chorioallantoic membrane assay and by subcutaneous inoculation of AIDS-KS cells into nude mice, which resulted in the development of angiogenic lesions composed of mouse cells and showing histological features similar to those of human KS lesions. These data suggest that AIDS-associated KS and possibly other types of KS may be initiated by signals that induce the growth of particular cells (spindle cells of lymphatic or vascular origin) and the expression of autocrine and paracrine activities.     

Hepa1-6细胞的最佳培养条件研究

[目的]探索Hepa1-6细胞的最佳培养条件。[方法]以小鼠Hepa1-6肝癌细胞为材料,采用正交试验设计研究RPMI1640和DMEM2种不同培养基及细胞接种密度、血清浓度、双抗浓度、D-Hanks漂洗次数、胰蛋白酶浓度和消化时间6个不同因素对Hepa1-6传代培养的影响;根据细胞在6个时间点的生长状况评分,筛选出Hepa1-6细胞的最佳培养条件。[结果]Hepa1-6细胞在含有20%血清的RPMI-1640培养基,接种密度为4×104个/cm2,100U/ml的双抗浓度,贴壁率在90%以上时,D-Hanks漂洗1次,0.5%胰蛋白酶消化2min,传代效果最好。[结论]通过正交设计筛选,为Hepa1-6细胞体外培养探索出最佳培养条件。     

Hepa1-6细胞的最佳培养条件研究

[目的]探索Hepa1-6细胞的最佳培养条件。[方法]以小鼠Hepa1-6肝癌细胞为材料,采用正交试验设计研究RPMI1640和DMEM2种不同培养基及细胞接种密度、血清浓度、双抗浓度、D-Hanks漂洗次数、胰蛋白酶浓度和消化时间6个不同因素对Hepa1-6传代培养的影响;根据细胞在6个时间点的生长状况评分,筛选出Hepa1-6细胞的最佳培养条件。[结果]Hepa1-6细胞在含有20%血清的RPMI-1640培养基,接种密度为4×104个/cm2,100U/ml的双抗浓度,贴壁率在90%以上时,D-Hanks漂洗1次,0.5%胰蛋白酶消化2min,传代效果最好。[结论]通过正交设计筛选,为Hepa1-6细胞体外培养探索出最佳培养条件。     

U1 small nuclear RNA genes are subject to dosage compensation in mouse cells

Multiple copies of a gene that encodes human U1 small nuclear RNA were introduced into mouse C127 cells with bovine papilloma virus as the vector. For some recombinant constructions, the human U1 gene copies were maintained extrachromosomally on the viral episome in an unrearranged fashion. The relative abundance of human and mouse U1 small nuclear RNA varied from one cell line to another, but in some lines human U1 RNA accounted for as much as one-third of the total U1. Regardless of the level of human U1 expression, the total amount of U1 RNA (both mouse and human) in each cell line was nearly the same relative to endogenous mouse 5S or U2 RNA. This result was obtained whether measurements were made of total cellular U1 or of only the U1 in small nuclear ribonucleoprotein particles that could be precipitated with antibody directed against the Sm antigen. The data suggest that the multigene families encoding mammalian U1 RNA are subject to some form of dosage compensation.     

PRODUCTION OF IMMUNITY TO DENGUE WITH VIRUS MODIFIED BY PROPAGATION IN MICE

It has been demonstrated that dengue virus can be propagated by intracerebral inoculation in mice. Although initial adaptation to the mouse is a tedious and difficult process, 16 consecutive passages have been achieved already in one series and further passages are in progress. The virus propagated in mice produced dengue in human volunters, but was not pathogenic for cotton rats, hamsters, guinea pigs or rabbits. Although it was evident that even after 2 serial passages in mice the virus produced a modified type of disease in human beings, tests with the 7th, 9th and 10th passage material indicated that the modification had become so marked that it could be used as a vaccine for the production of immunity against dengue.