大鼠卵黄囊的分化及其肿瘤性转化研究

目的研究12d大鼠胚胎脏层卵黄囊(VYS)向多胚层组织分化的潜能和在逆转录病毒感染下的肿瘤性转化特征。方法在不同培养条件、移植位点的条件下,观察VYS体内外分化的改变;另外利用逆转录病毒载体将荧光蛋白基因(GFP)转染12d卵黄囊细胞,对GFP标记的转化细胞进行体内外研究。结果在不同培养条件下,均对体外培养的或体内移植的大鼠卵黄囊向三个胚层分化的进程无特异的导向性。将荧光蛋白标记卵黄囊克隆细胞接种在裸鼠皮下长出了未分化的间质细胞肉瘤。结论12d大鼠胚胎脏层卵黄囊具有向三胚层分化的潜能;逆转录病毒感染导致卵黄囊间质细胞发生肿瘤性转化。     

冻融对温带土壤可溶性氮库、氮转化过程及细菌群落多样性的影响

土壤冻融会影响土壤氮的有效性。氮的转化与土壤微生物密不可分,而土壤冻融对温带土壤细菌群落的影响并不十分清楚。假设: 冻融影响细菌群落结构多样性及其组成,从而改变土壤可溶氮含量和氮转化过程。为了验证这一假设,本研究设计了不同冻融循环次数(分别为6次和15次循环)的培养试验,并以2 ℃恒温培养作为对照。结果表明: 随着冻融循环次数的增加,可溶性全氮、可溶性无机氮、微生物生物量氮和净氮矿化率均显著降低。冻融循环次数对细菌α多样性(包括Chao1和Shannon指数)无显著影响,恒温培养的培养周期数与细菌的α多样性呈显著正相关。冻融处理显著影响细菌群落功能和组成,但冻融循环次数对细菌群落结构的影响较小。偏冗余分析表明,冻融处理下细菌群落结构和功能多样性与土壤可溶氮含量和氮转化过程密切相关。     

大肠杆菌JM109感受态形成因素分析

目的:分析大肠杆菌JM109感受态形成因素,提高转化效率。方法:采用不同生长状态、不同转化溶液、不同保存时间及热激处理时间的细菌制备感受态,分析转化效率。结果:以20mmol/L MgCl2 80mmol/L CaCl2为处理液,经活化培养OD600为0.82的菌液制备感受态细胞,4℃放置12~24h之内,42℃热激处理60s,转化效率最高,可达9.8×106~1.2×107cfu/μg DNA(pUC19)。随着质粒长度增加,转化效率下降。结论:感受态细胞形成与生长状态关系密切,金属离子、有机溶剂对感受态的形成影响显著。感受态形成过程中,细胞可能发生了一系列的生理变化。     

中华大蟾蜍(Bufo bufo gagarizans)血淋巴细胞的体外培养及其转化

本文报道了中华大蟾蜍血淋巴细胞的培养及其在植物血球凝集素(PHA)的刺激下所引起的转化,并初步查明了转化的淋巴细胞的S期、G_2期所占时间。用氚标记放射自显影手段测定了细胞的转化率。用姐妹染色单体区分着色的方法决定细胞的分裂次数。实验结果表明,在新鲜血液中有0.1—0.2％的白细胞具有合成DNA的能力。培养3天后,淋巴细胞转化率为14.8％,第5天达48％。培养的第6天有6—10％为第2次分裂。在秋水仙素处理3小时的情况下,有丝分裂指数为7％,延长处理时间至10—15小时,则平均达10％。最高有丝分裂指数为29％。转化淋巴细胞的S期为16小时,G_2期为3小时。     

不同离子剂量介导外源薄荷全DNA转化对拟南芥菜生长发育的影响

在离子束介导外源全DNA转化中,外源DNA包含有供体的所有遗传信息,对受体的影响是多方面的,因此转化效果无法用统一的转化率来衡量,这也增加了转化过程中最佳转化条件的选择难度。本论文的主要工作是根据不同离子剂量介导外源全DNA转化的拟南芥菜在遗传和生理上的变化选择合适的转化剂量。剂量实验结果表明:拟南芥菜种子的剂量-存活率曲线为"马鞍型",在"鞍桥"上选择0.5×1017ions/cm2、1.5×1017ions/cm2和2.5×1017ions/cm2三个有代表性的剂量介导薄荷全DNA转化拟南芥菜,三个转化当代群体的出芽、成苗长势以及表型变异情况有明显的不同,综合分析表明:1.5×1017ions/cm2为离子束介导外源全DNA转化拟南芥菜的合适剂量。     

不同培养时期的大肠杆菌和枯草芽孢杆菌间发生的跨属自然遗传转化

携带穿梭质粒的大肠杆菌与作为受体的枯草芽孢杆菌分别培养至不同生长阶段混合均匀后静置40min,涂布选择性平板,37℃培养30h后得到一定数目的转化子,DNaseⅠ敏感实验证实质粒是通过自然遗传转化而非其它形式发生转移。实验发现大肠杆菌可以在特定生长时期向胞外分泌DNA,并且在对数期具有最高的提供质粒的能力,而生长后期的细胞因为体系中DNase量的增加转化频率下降。进一步的研究发现枯草芽孢杆菌在营养丰富的LB培养基中也具有与基本培养基中相当的转化能力,并且在对数生长前期具有较高的转化频率。     

常压低温等离子体对羊肚菌培养基质的杀菌效果及工艺优化

以羊肚菌培养基质为试验对象,采用常压低温等离子体技术对其进行杀菌处理,分别研究了等离子体处理过程中电压、频率、时间和次数4种单因素变量对菌落数和水分的影响,并通过正交实验优化了处理工艺条件。结果表明,细菌和真菌的杀菌率随常压等离子体处理电压、处理时间和处理次数的增大而增加,水分损失也随之增加,而随着频率的增大,杀菌率呈现先增大后减小的“火山型”趋势。不同处理条件下的杀菌效果普遍存在显著性差异(P<0.05)。正交实验表明4种影响因素大小为：频率>电压>时间>次数,最佳杀菌工艺条件为：电压30kV,频率9.6kHz,时间45 min,次数1次,此条件下细菌和真菌杀菌率分别为96.87%和93.77%,且水分损失较少。     

光质对石刁柏愈伤组织培养中生长和过氧化物酶的影响

报道了在石刁柏愈伤组织培养中,采用５种不同光质处理,对愈伤组织生长及其过氧化物酶（ＰＯＤ）活性及同工酶的影响,实验表明,不同光质处理对生长有不同的效应,蓝光、红光下的培养物生长较快。不同光质对培养物质的ＰＯＤ活性亦有不同效应,ＰＯＤ同工酶谱亦有不同,蓝光、红光下的培养物,ＰＯＤ活性较高。ＰＯＤ活性变化与愈伤组织生长呈正相关。     

药物安全性评价中豚鼠全身过敏试验方法的研究

目的在新药临床前安全性评价中对药物致敏性的研究日益受到重视。本实验基于我国新药评价指导原则,分析探讨SFDA颁布的指导原则中豚鼠全身过敏实验方案中可能影响实验结果的问题和因素。方法在GLP条件下,以体重为300～400克的豚鼠为实验对象,以牛血清白蛋白、生理盐水等受试物为对照,研究不同致敏剂量、激发剂量、激发时间、激发途径和激发次数等条件下,豚鼠全身过敏的发生情况。结果在一定剂量范围内,豚鼠全身过敏反应的发生率与致敏剂量、激发剂量和激发途径无关,但症状发生的严重程度与致敏剂量、激发剂量和激发途径有关。结论新药过敏性评价研究应参照指导原则,注意试验的剂量设计和给药途径。     

催乳素对培养的绵羊睾丸间质细胞睾酮分泌的影响

在绵羊睾丸间质细胞体外无血清长期培养的条件下,研究了催乳素对睾丸间质细胞睾酮分泌的调节作用。实验结果表明,催乳素可增强细胞对人绒毛膜促性腺激素(hCG)刺激的反应。催乳素的这种作用呈双相调节。睾酮分泌量显著高于hCG和催乳素单独作用时的总和。在hCG存在下,不同的底物转化为睾酮的量不同。其中雄烯二酮和孕酮转化为睾酮的方式存在着双相性。脱氢表雄酮转为睾酮的量少,不存在双相性,而与其剂量成正比。催乳素在hCG存在下可调节底物转化为睾酮。低剂量的催乳素(1ng/ml)可使一定剂量的孕酮(10～30ng/ml)转化为睾酮的量明显增加,而高剂量的催乳素(>10ng/ml)却明显地抑制孕酮转化为睾酮。催乳素可明显地抑制雄烯二酮转化为睾酮,与剂量无关。可见催乳素对于孕酮和雄烯二酮这两个关键底物转化为睾酮的调节是不同的。催乳素增强hCG刺激睾酮分泌的作用可能部分是通过其促进孕酮转化为睾酮来实现的。     

X-ray induction of O6-alkylguanine-DNA alkyltransferase protects against some of the biological effects of N-methyl-N'-nitro-N-nitrosoguanidine in C3H 10T1/2 cells

We have shown previously that the repair of O6-methylguanine can be induced in murine fibroblasts (C3H 10T1/2 cells) by exposure to X rays. The magnitude of the response is less, however, than is observed in the well-characterized adaptive response of various prokaryotes to methylating agents. To determine whether the induction of O6-alkylguanine-DNA alkyltransferase in C3H 10T1/2 cells is sufficient for protection against the genotoxic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were challenged with MNNG after alkyltransferase induction by 1.5 Gy X rays and assayed for cytotoxicity, mutagenicity, and neoplastic transformation. Preirradiated cells were significantly more resistant to the mutagenic effects of MNNG as scored by formation of ouabain-resistant colonies. The protective effect was greatest in cells challenged with a low dose (0.2 or 0.4 micrograms/ml) of MNNG. Protection against neoplastic transformation by MNNG was also observed, although the protective effect in this case was significant only in cells treated with a high dose (1.0 micrograms/ml) of MNNG. In cells that were preirradiated, there was no reduction in the cytotoxicity caused by MNNG or the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These data indicate that alkyltransferase induction in C3H 10T1/2 cells is sufficient to protect cells against some of the genotoxic effects of the alkylating agent MNNG. The data also suggest that formation of O6-alkylguanine may not be the only means by which alkylating agents can transform C3H 10T1/2 cells.     

Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.     

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.     

Chemical carcinogen alteration of SV40 virus induced transformation of normal human cell populations in vitro.

The frequency of simian papovirus 40 (SV40) induced transformation of human cells was enhanced after pretreatment with either napthylamine-2,N-methyl-N'-nitrosoguanidine (MNNG), N-acetyl-2-fluorenylacetamide (N-Ac-AAF), benzo[a]pyrene (BP), aflatoxin B1, propane sultone (PS), beta-propiolactone, 4-nitroquinoline oxide (4-NQO), methylmethane sulfonate (MMS) or diethyl nitrosamine (DEN). Posttreatment with 4-NQO, MMS, MNNG or DEN inhibited transformation; while posttreatment with either aflatoxin B1, beta-propiolactone or napthylamine-2 did not alter transformation similar to the action of N-Ac-AAF and BP. All carcinogens that altered transformation after pretreatment damaged cellular DNA. Pretreatment or posttreatment with carcinogens 3-methylcholanthrene (3-MCA) or 7,12-dimethylbenzanthrene (7,12-DMBA), that did not damage cellular DNA also did not enhance transformation. Moreover, pre- or posttreatment with other weak or non-carcinogens that did not damage cellular DNA did not alter virus induced transformation. All foci formed in the co-carcinogen treated cultures whether the carcinogen inhibited or enhanced transformation were virus directed. While a similar pattern of response existed for carcinogens that either enhance or inhibit transformation, each of the carcinogens that enhanced or inhibited foci formation damaged cellular DNA. Moreover, those carcinogens that enhanced focus formation, compared to the carcinogens that inhibited focus formation, exhibited similar DNA damage profiles.     

Absence of various manifestations of adaptive response in Bacillus subtilis transformants

Competent cells of Bacillus subtilis are more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) than total population, exhibit higher level of spontaneous mutations to kanamycin resistance. However, the absolute number of mutated transformants doesn't rise with MNNG treatment in the range of 5 to 50 micrograms per ml. The adaptation to low concentrations of MNNG affects neither spontaneous nor MNNG-induced mutagenesis in the competent (transformed) cells, in contrast to their resistance which is stronger for the adapted transformants. The transformation by MNNG-treated plasmid pUB 110 doesn't reveal any difference between adapted and non-adapted cultures in transformation efficiency decline.     

Ultrastructural changes in rat colorectal epithelium and tumors after treatment with N-methyl-N'-nitro-N-nitrosoguanidine and secondary bile acids.

Transmission electron microscopy (TEM) was used to study ultrastructural changes that accompanied the tumorous transformation of the descending rat colon epithelial cells, following short treatment with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), with subsequent prolonged treatment with secondary bile acids, lithocholic (LCA) and deoxycholic (DCA), which enhanced tumor formation. Colon epithelial cells after long treatment with bile acids alone were characterized by the presence of an irregular nuclear membrane, ring-shaped rough endoplasmic reticulum (RER), collagen-like tonofilaments and membrane-bound mucous vacuoles. Tumor cells which developed following treatment with MNNG alone were characterized by the irregular shape of the nuclear membrane and, sometimes, by polynuclei, accumulation of large amounts of mitochondria, loss of cell-cell contacts and by endocytosis of the cell membrane. After combined treatment with MNNG and LCA, many mitochondria lost their membranous envelope; in the cytoplasm many collagen-like tonofilaments, ring-shaped RER and many free ribosomes were present. After treatment with MNNG and DCA, many polysomes were found in the cytoplasm. It was apparent that treatment with MNNG alone caused the development of adenocarcinoma-like tumors, while additional treatment with secondary bile acids significantly enhanced these changes, which were accompanied by the development of atypia and anaplasia of epithelial cells, with many irregularities in intracellular organization.     

Enhancement of plasminogen activator activities by N-methyl-N'-nitro-N-nitrosoguanidine in tuberous sclerosis fibroblasts

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) enhanced the levels of plasminogen activator (PA) activity in fibroblast cells derived from the skin of patients with tuberous sclerosis. The enhanced enzyme levels were not correlated with those of cloning efficiency nor those of DNA synthesis after MNNG treatment. Enzyme enhancement was also observed in fibroblasts of ataxia telangiectasia and in human neoplastic glia cells, but not in fibroblasts of normal children. The PA induction test may be sufficiently sensitive for the detection of the cellular defects of tuberous sclerosis.     

Chemical oncogenesis in cultured mouse embryo cells in relation to the cell cycle.

Using the C3H/10T 1/2 CL8 line of mouse embryo fibroblasts and three different methods of obtaining cell cycle synchrony, namely arginine or isoleucine deficiency and release from postconfluence inhibition of growth, a sensitive phase for oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been found. This sensitive phase is located somewhere between the G1/S boundary and a point 4 hr prior to this marker. Methylation of cellular macromolecules by tritiated MNNG is not cycle-dependent in cells synchronized by arginine deficiency. The capacity of cells to repair DNA single strand breaks produced by MNNG was examined by alkaline sucrose sedimentation analysis in cells synchronized by arginine deficiency and treated with MNNG during phases of the cell cycle sensitive and insensitive to oncogenic transformation. Whereas DNA repair was found to be equally rapid in cells treated just before S phase (I), or just after commencement of DNA synthesis (III), transformation was maximal in I. By contrast, cells treated when blocked by arginine deficiency (II) repaired DNA slowly and were not sensitive to malignant transformation. Cells in I and II, which repaired DNA at very different rates, were equally sensitive to MNNG-induced lethality, while cells in III, which repaired DNA at the same rate as cells in I, suffered greater lethality. Thus, in this system it was concluded that there was no direct correlation between DNA repair, as measured by alkaline sucrose sedimentation analysis of prelabeled DNA, and malignant transformation or lethality produced by MNNG. In preliminary experiments malignant transformation induced by cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) has been found to occur mainly in S phase, indicating that diverse chemical oncogens may have different sites of action, or that activation of chemical oncogens is cell cycle-specific for some agents.     

Chemical and viral transformation of cultured skin fibroblasts from patients with familial polyposis coli

Treatment of skin fibroblasts from an FPC patient with 4NQO or MNNG followed by sequential passaging caused morphological changes of the cells, which showed characteristics of transformed cells such as a high frequency of colony formation in agarose, increased growth ability, and chromosomal abnormalities. This and other fibroblast lines from 5 of 12 FPC patients had an increased susceptibility to 4NQO cytotoxicity, which was caused by enhanced 4NQO-reductase activity rather than by reduced DNA repair. However, the susceptibility to cytotoxicity of MNNG and repair of MNNG-damaged DNA were normal in FPC cells. The tumor promoters TPA and DHTB enhanced the frequency of chemical transformation of the FPC fibroblasts, and protease inhibitors suppressed the promoter-enhanced transformation. The skin fibroblasts from many FPC patients exhibited increased susceptibility to transformation by murine sarcoma viruses. Analysis of the viral DNA and RNA after infection revealed that the increased susceptibility is determined at an early stage of transformation. Two out of 5 MNNG-transformed clones of FPC fibroblasts, isolated from agarose, had increased expression of c-Ki-ras or c-Ha-ras, and 4 of 4 MSV-transformed clones showed high expression of viral Ki-ras. These clones grew further after isolation from agarose, but were mortal and did not form tumors in nude mice. The present results suggest that additional changes in morphologically transformed FPC fibroblasts are required for malignant transformation.     

Chromosomal aberration,mutation and morphological transformation of Syrian hamster embryonic cells after exposure to methylnitrosocyanamide

Hamster embryonic fibroblasts were treated directly with various concentrations of methylnitrosocyanamide (MNC), a nitrosated product of methylguanidine (MG) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Then they were examined for chromosomal aberrations, morphological transformation and mutations resistant to 8-azaguanine (8AG) and 6-thioguanine (6TG). Direct treatment with 2 to 10 × 10^?6 M MNC caused a marked, dose-dependent appearance of 8AG- and 6TG-resistant mutations. The ability of MNC to induce mutations was similar to that of MNNG. Cultured embryonic fibroblasts in metaphase plates also showed a marked dose-dependent increase in chromosomal aberrations within 24 h after direct treatment with MNC of MNNG. Moreover, MNC and MNNG caused similar rates of morphological transformation.