凝血酶诱发人支气管上皮细胞恶性转化

目的探讨凝血酶对人类支气管上皮细胞的致癌性和转化涉及的相关机制。方法用75nmo·lL-1凝血酶持续刺激人类乳头状瘤-18永生化的人支气管上皮细胞(BEP2D)70d,观察不同代龄细胞的血清抗性、软琼脂克隆形成率,流式细胞术观察细胞周期改变,逆转录-聚合酶链反应检测凝血酶受体-蛋白酶活化受体(PAR1),c-myc和p21~(cip1)基因表达水平,蛋白质免疫印迹检测C-MYC和P21~(cip1)蛋白表达变化,鉴定细胞的恶性转化表型。结果经传代培养,凝血酶处理的第10代BEP2D细胞血清抗性增强,接种效率提高,第20代细胞呈非贴壁依赖性生长,软琼脂克隆形成率升高达(0.461±0.009)%。流式细胞术结果显示,第22代细胞处于S期比例为55.63%。凝血酶转化第25代、第27代细胞PAR1基因、c-myc基因及C-MYC蛋白表达水平上调。相反,p21~(cip1)基因和P21~(cip1)蛋白表达减弱。凝血酶处理的第20代细胞已具有较为稳定的转化表型和细胞生物学特性,可能是具有裸小鼠致瘤型的恶性转化细胞。每升2×104抗凝血酶单位水蛭素能够拮抗凝血酶对BEP2D细胞的恶性转化作用。结论凝血酶对人支气管上皮细胞具有恶性转化作用,c-myc基因转录和蛋白表达活化与p21~(cip1)失活在这一过程中发挥关键作用。     

人表皮干细胞体外分离和培养的研究

目的建立人表皮干细胞体外分离和培养的技术方法。方法用DispaseⅡ酶和胰蛋白酶两步法分离出角朊细胞和成纤维细胞,并以人成纤维细胞条件培养基为基础制备表皮干细胞培养液,用以人表皮干细胞(Ⅳ型胶原快速粘附法富集分离)的体外培养,通过检测β1整合素和角蛋白19的表达水平及其克隆形成率和克隆维持时间对其进行鉴定,角朊细胞作对照。结果表皮干细胞体外培养,可见细胞呈克隆样生长、β1整合素及角蛋白19免疫组织化学染色呈阳性,且其克隆形成率和克隆维持时间分别为(17.04±1.01%)和15～18d,明显高于对照组的(8.72±0.73%)和9～10d(P<0.01)。结论应用Ⅳ型胶原快速粘附法及人成纤维细胞条件培养基,对人表皮干细胞成功进行了体外分离和培养,为表皮干细胞体外的大量扩增奠定了基础。     

人表皮干细胞的分离培养

目的 探索人表皮干细胞原代培养方法.方法 将皮肤标本进行分离,用DispaseⅡ酶消化表皮皮片后,重悬细胞,接种于铺有Ⅳ型胶原的培养瓶进行培养.表皮干细胞的鉴定采用免疫细胞化学技术检测其表面标记物β1整合素、CK19的表达.对表皮干细胞与角质形成细胞的克隆形成情况进行比较.结果 倒置相差显微镜下观察细胞形态及生长状况良好,表皮干细胞β1整合素及CK19角蛋白染色阳性.表皮干细胞克隆形成率高于角质形成细胞（P＜0.05）.结论 采用本方法进行人表皮干细胞的培养较为理想.     

研究表皮黑素细胞与毛囊角质形成细胞在接触性共培养体系中的相互作用

目的研究分析表皮黑素细胞和毛囊角质形成细胞在接触性共培养体系的建立,以及相互作用。方法用胰酶融化游离毛囊与包皮的表皮,获得纯度角度高的角质形成细胞与黑素细胞,第三代毛囊角质形成细胞在6孔板中接种,2 d以后按照表皮黑素细胞与毛囊角质形成细胞之间的比例,按照1∶2、1∶10、1∶20的比例接种黑素细胞,其中在1∶10的比例中,加入-黑素细胞刺激素,在干预6 d以后,用NKI/beteb作一抗。结果以1∶10的接种比例,很符合表皮基底层的黑素细胞与角质形成细胞比例;但是在1∶2情况下可获得数量多的表皮黑素细胞。而在1∶20情况下可以发现角质细胞、黑素细胞之间的联系。结论表皮黑素细胞与毛囊角质形成细胞在接触性共培养体系下,非常适合用于人色素系统方面的研究。     

p16基因缺失在成人ph阴性急性B淋巴细胞白血病中的临床意义

目的 探讨 p16基因缺失对成人 Ph阴性急性 B淋巴细胞白血病（B-ALL）患者的临床特征及预后的影响。方法 回顾性分析210例初发成人ph阴性B-ALL患者的临床资料,根据初发时检测的p16基因状态分为p16基因缺失组 61例和无缺失组 149例。比较 p16基因缺失与无缺失组患者的临床、免疫表型、细胞遗传学、分子学特征及预后。结果 p16基因缺失组中伴 CD20表达者所占比例明显高于无缺失组（47.5% vs. 30.8%,χ2=5.238,P＜0.05）,2组患者间其余免疫表型和细胞遗传学分布差异无统计学意义。p16基因缺失组造血干细胞移植率和完全缓解率与无缺失组差异均无统计学意义,复发率明显高于无缺失组（χ2=12.027,P＜0.05）。79例复发患者中,4例患者初发时未检测出 p16 基因缺失,而复发时检测出 p16 基因缺失。p16 基因缺失组患者的 OS 和 DFS 明显低于无缺失组（Logrank χ2分别为 16.715、21.237,均 P＜0.05）。p16基因缺失组中接受造血干细胞移植患者的 OS和 DFS均优于化疗患者（Log-rank χ2分别为25.316、20.637,均 P＜0.05）。p16基因缺失组CD20阳性患者OS和DFS均明显低于CD20阴性者（Log-rank χ2分别为 7.782、5.733,均 P＜0.05）。结论 p16基因缺失患者 CD20阳性表达率高且预后差,造血干细胞移植能够明显改善p16基因缺失患者的预后。     

重组pAd/CMV/V5-DEST-p16载体的构建及其对人肝癌细胞的抑制作用

目的构建pAd/CMV/V5-DEST-p16重组腺病毒载体,并观察野生型p16基因对人肝癌细胞SMMC7721株的抑制作用。方法合成人p16-INK4表达基因。构建pENTR 1A-p16质粒。通过LR反应,入口克隆pENTR 1A-p16质粒的外源性合成的修饰后的p16 cDNA,取代目的载体pAd/CMV/V5-DEST中的ccdB基因,形成表达克隆pAd/CMV/V5-DEST-p16。测序证实质粒含有目的基因。PacI酶切后的重组腺病毒载体,通过脂质体2000介导,转染293A细胞,产生缺陷性的重组腺病毒。W estern blot分析显示在由重组腺病毒介导的野生型p16基因在肝癌细胞SMMC7721中能够表达蛋白。被感染的细胞的生长受抑制。结果构建了重组p16腺病毒载体,产生缺陷性的重组腺病毒,野生型p16基因对人肝癌细胞SMMC7721株有抑制作用。结论用通路克隆系统构建重组p16腺病毒载体稳定、可靠、方便,腺病毒能够介导野生型p16基因在肝癌细胞中表达,并抑制细胞的生长。     

碱性成纤维细胞因子对SD大鼠表皮干细胞分化为神经干细胞的影响

目的 研究碱性成纤维细胞因子(bFGF)对大鼠表皮干细胞分化为神经细胞的影响。 方法 分离获得饲养了1~3天的新生SD大鼠表皮基底层组织,利用10 min快速贴壁法消化、分离获得表皮干细胞,于倒置显微镜下观察表皮干细胞的形态。培养表皮干细胞的培养基为K-SFM,然后按照不同密度表皮干细胞进行分组处理(0.1×10⁷/mL,0.3×10⁷/mL,0.5×10⁷/mL,0.1×10⁶/mL),每组加入20 ng/mL的bFGF,利用细胞免疫组织化学法检测细胞标志物Nestin和NSE的变化,以及观察细胞形态发生的变化。 结果 成功分离得到SD大鼠表皮干细胞;bFGF诱导后,0.3×10⁷/mL组和0.1×10⁷/mL组细胞第3天即可见细胞开始伸展生长,在约1周时细胞开始分化成神经细胞;且在细胞形态发生双极化改变的数目趋势上也具有一致性;Nestin和NSE检测均呈阳性表达。     

不同剂量137Csγ-射线辐射后造血干/祖细胞辐射敏感性差异研究

摘要：目的 探讨不同剂量137Csγ-射线照射后不同时间点C57 BL/6小鼠造血细胞放射敏感性的差异。方法 选取6~8周龄雄性C57BL/6小鼠72只,完全随机法分为对照组和照射组。照射组小鼠分别接受2、4、6 Gy137Csγ射线一次性全身照射,对照组小鼠接受假照射。小鼠分别于受照后14 d、35d和56d断颈处死,取外周血进行血象测定,取骨髓细胞测定有核细胞数目和造血干/祖细胞数目。结果 不同剂量照射后小鼠的外周血常规指标有明显变化,白细胞数目明显下降,其次是血小板数目,且具有剂量效应关系;在照射后14d,2Gy、4Gy和6Gy照射组小鼠骨髓有核细胞数目与对照组比较分别下降21.9%、39.9%和54.4%（t=4.311、6.401、8.007,P<0.05）;照射后35d和56d,6Gy照射组小鼠骨髓有核细胞和造血祖细胞数目显著低于对照组（t=4.185、3.596,P<0.05）。照射后14d、35d和56d,2Gy、4Gy和6Gy照射组小鼠骨髓造血干细胞数目持续低于对照组（t=9.706、3.427~7.465,P<0.05）。结论 不同剂量137Csγ-射线照射对小鼠造血系统造成不同程度的损伤,造血祖细胞较造血干细胞辐射敏感,且辐射对造血干细胞造成的损伤是持久性的。     

应用CRISPR/Cas9系统在G401细胞株中敲除p21基因

目的 运用 CRISPR/Cas9 基因编辑技术, 在人恶性横纹肌样瘤细胞株 G401 中敲除 p21 基因。 方法 通过反转录定量 PCR（RT-qPCR）及 Western blot 检测各瘤细胞株中 p21 的表达, 针对 p21 基因作用的功能域, 设计了靶向人 p21 基因第 3 个外显子的向导 RNA（sgRNA）, 克隆入 lentiCRISPR v2 载体。 将测序及酶切鉴定正确的重组质粒在 293T 工具细胞中制备慢病毒颗粒并感染 G401 细胞, 使用嘌呤霉素进行阳性细胞筛选, 显微镜下挑取单克隆细胞团并继续培养获得 G401 单克隆细胞株。 提取单克隆细胞株 RNA 及蛋白, 利用 RT-qPCR 及 Western blot 方法检测细胞株中 p21 的敲除效果。 结果 p21 在人横纹肌样瘤细胞中高表达。 成功构建靶向 p21 基因的 lentiCRISPR v2-sgRNA 重组慢病毒质粒。 与对照组相比, 筛选得到的 G401 亚克隆细胞系中 p21 蛋白表达缺失。 结论 针对难转染的 G401 细胞, 应用 CRISPR/Cas9 系统成功构建了 p21 基因敲除的稳定株, 为后续深入研究 p21 在人恶性横纹肌样瘤中的作用机制奠定了基础。     

SLE患者血清对小鼠BMMSC生长、增殖及凋亡的影响

目的 了解小鼠骨髓间充质干细胞(BMMSC)生长增殖特性,探讨系统性红斑狼疮(SLE)患者血清对其影响.方法 分离培养BALB/c小鼠第二代BMMSC,加入不同浓度SLE患者血清,分别在第2,4,6,8天进行细胞计数,描绘生长曲线;第二代培养第6天的BMMSC加入不同浓度SLE患者血清,MTT法测定细胞增殖情况,Annexin V/PI测定细胞凋亡.结果 正常人血清组对BMMSC增殖、凋亡与对照组无明显差别;小鼠第二代BMMSC生长在第6天达到高峰;5%、10%或15%SLE血清均对BMMSC生长呈浓度依赖性抑制作用,可增加BMMSC的凋亡.结论 SLE患者血清中可能存在某些物质抑制BMMSC的生长.     

Minimal influence of metallothionein over-expression on nickel carcinogenesis in mice

Metallothionein (MT) is a metal-binding protein associated with tolerance to metals and oxidative stress. Nickel is a metal carcinogen potentially acting through oxidative attack on critical biomolecules. We investigated the role of MT in nickel carcinogenesis using MT-transgenic mice that constitutively over-express MT-I in all tissues tested. Groups of 25 male MT-transgenic and wild type (C57BL/6; WT) mice received intramuscular injections of nickel subsulfide (Ni3S2) in both thighs at doses of 0 (control), 0.5, or 1.0 mg/site at 12 weeks of age and were observed for 104 weeks. Injection site tumors (ISTs; primarily fibrosarcomas) started occurring 45 weeks after nickel injection and IST incidence was similar in the WT (control - 0%, 0.5 mg/site - 20%, 1.0 mg/site - 40%) and MT-transgenic mice (control - 0%, 0.5mg/site - 28%, 1.0mg/site - 29%.). At the 0.5 mg/site dose the average time to IST in MT-transgenic mice was approximately 13 weeks shorter than in WT mice. Spontaneous lung tumors developed in 25% of control WT mice but none developed in control MT-transgenic mice. A nickel dose-related trend for increased lung tumors occurred in MT-transgenic mice but not in WT mice. Thus, the over-expression of MT did not significantly mitigate the carcinogenic response to nickel.     

Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system

Exfoliated human urinary tract epithelial cells and renal tubular cells from urinary sediments of healthy adults, of urological patients and of internal patients were isolated and cultured. Cells started proliferating within 1 week after seeding a sediment. Proliferating cells formed colonies of different morphologies, designated as type-1 or type-2 cell colonies. Type-1 cell colonies showed irregular contours and spindle-like cells within the colonies. Subcultivation of type-1 cells for up to six passages was possible. Type-2 cell colonies showed smooth-edged contours and subcultivation was not possible. The epithelial character of type-1 cells was demonstrated by positive immunohistochemical staining for cytokeratin-7. In contrast to carbonic anhydrase-positive stained Madin Darby canine kidney cells (MDCK), which were used as positive controls for renal tubular cells, type-1 cells were carbonic anhydrase-negative on staining with the cobalt phosphate method. This indicates that type-1 cells were not of renal tubular origin. Type-2 cells were positively stained for carbonic anhydrase, indicating that type-2 cells were renal tubular cells. Type-2 cell colonies could be assigned to two subgroups with different cell forms. Colonies of cobblestone-like cells more often occurred than type-2 cell colonies with spindle-like cells, which are described in this study for the first time. Colonies with cobblestone-like cells formed domes (hemicysts), whereas spindle-like type-2 cell colonies did not. Cultures of urinary sediments from healthy adults, elderly multimorbid patients treated with furosemide, and urological patients with urolithiasis treated with sulfamethoxazole/trimethoprim and/or with a percutaneous nephrostomy catheter were compared. In 52% of all cultured sediments from healthy adults, in 30% of those from multimorbid patients, and in 75-80% of those from urological patients cells proliferated to colonies. The ratios of type-1 to type-2 cell colonies were 3.3:1 (healthy adults), 1.4:1 (urological patients with urolithiasis), and 1.8:1 (urological patients with urolithiasis, urine was directly collected from the renal pelvis with a percutaneous nephrostomy catheter). Successful cultures of the urinary sediments from these three groups revealed means of 3 or 4 colonies, 14 colonies, and 21 colonies, respectively. Differences in the number of colonies in relation to sex were observed only for the group of urological patients. It was shown that type-1 cells were urothelial cells, which did not show morphological differences due to their locations of origin within the urinary tract, whereas type-2 cells were probably renal tubular cells. These findings offer new aspects in the culturing of human urothelial or kidney epithelial cells with a method based on noninvasive collecting of specimens and requiring only minimal culture effort. The cultures obtained by this method can be used for in vitro studies in toxicological and clinical research.     

Pathogenecity of fosfomycin-resistant strains isolated from Escherichia coli

Two fosfomycin-resistant strains, FRC14 (parent strain, Escherichia coli [E.coli] c73-18) and FRK104 (parent strain, E. coli O124), were isolated from spleens before the bacterial disappearance, after inoculating the parent strains intraperitoneally into mice and treating them with a single oral dose of fosfomycin. The resistant strains were successfully isolated by a replica method from a mass of sensitive cells of respective parent. To elucidate the pathogenesis of the resistant strains, their characteristics were investigated. The MIC of fosfomycin for FRC14 was 25 micrograms/ml (4 times the MIC for the parent) and that for FRK104 was 100 micrograms/ml (8 times the MIC for the parent). The strain FRC14 showed a defective utilization of sn-glycerol 3-phosphate (G3P), but utilization of other carbohydrates was similar to that of the parent strain. Thus, the strain FRC14 seemed to be a glpT mutant. The strain FRK104 did not use variety of carbohydrates including G3P, but used glucose 6-phosphate. The utilization of G3P was recovered in the presence of cAMP. Thus, the strain FRK104 seemed to be a ptsI mutant. These resistant strains were diminished their killing activity for mice in comparison to that of the each parent strain when they were inoculated intraperitoneally. The cell number of FRC14 decreased or disappeared in blood and spleen in mice, while that of the parent increased. The strain FRK104 diminished its ability of producing keratoconjuctivitis in guinea pigs in comparison to that of the parent strain.     

Taxol normalizes the impaired agonist-induced beta2-adrenoceptor internalization in splenocytes from GRK2+/- mice

G protein-coupled receptor kinase 2 (GRK2) is involved in the agonist-induced desensitization of beta2-adrenoceptors. In addition, GRK2 is capable of binding and phosphorylating tubulin. Interestingly, microtubule dynamics profoundly affect agonist-induced internalization of beta2-adrenoceptors. Here, we analyzed agonist-induced beta2-adrenoceptor internalization and signaling in splenocytes from GRK2+/- mice that have a approximately 50% lower level of GRK2 protein compared to wild type (WT) mice. In addition, we investigated the role of microtubule stability in these processes. Splenocytes from GRK2+/- mice express approximately 50% less beta2-adrenoceptors on the cell surface and show impaired agonist-induced beta2-adrenoceptor internalization. Disruption of microtubules using colchicine reduces agonist-induced beta2-adrenoceptor internalization in cells from WT, but not in cells from GRK2+/- mice. Importantly, increasing tubulin stability by taxol almost completely restores the defective agonist-induced beta2-adrenoceptor internalization in cells from GRK2+/- animals, without affecting WT cells. Despite lower surface receptor numbers, cells of GRK2+/- mice show normal beta2-adrenoceptor agonist-induced cAMP responses. Although interfering with microtubule stability has major effects on agonist-induced receptor internalization in GRK2+/- cells, microtubule dynamics do not influence cAMP responses. Our data suggest that cells with low GRK2 adapt to the lower GRK2 level by decreasing the number of beta2-adrenoceptors on the cell surface. In addition, the cellular GRK2 level determines the extent of agonist-induced beta2-adrenoceptor internalization via a mechanism involving microtubule stability. Importantly, however, normalization of agonist-induced receptor internalization by taxol is not sufficient to alter receptor signaling.     

核糖体蛋白S6激酶1抑制剂对气道上皮干/祖细胞 增殖及分化功能的影响

李敏敏 1,李宽 1,2,孙昕 1,2,吴琦 1,2,陈怀永 摘要：目的 研究mTOR信号通路下游元件核糖体蛋白S6激酶1（S6K1）抑制剂PF-4708671对气道干/祖细胞增 殖与分化功能的影响。方法 准备10只C57BL/6小鼠,采用流式细胞分选技术将气道干细胞（vClub）和气道祖细胞 （Club）从小鼠气道分选出来;采用类器官培养技术分别把vClub细胞、Club细胞和成纤维细胞（MLg）混合培养。细胞 设0、4、20、100 nmol/L PF-4708671处理组,培养第8天时显微镜下观察克隆生长情况,统计克隆形成数量;第10天时 荧光定量PCR检测S6K1激酶基因Rps6kb1、Club细胞标志物细胞色素氧化酶（Cyp2f2）、纤毛细胞标志物乙酰化微管 蛋白（Act）和叉头框转录因子J1（Fox j1）,杯状细胞标志物氯化物通道钙活化家族成员3（Clca3）叉头框转录因子a3 （Foxa3）的mRNA表达情况。结果 不同浓度的PF-4708671对vClub细胞克隆数量无明显影响（P＞0.05）,而4、20、 100 nmol/L PF-4708671浓度组Club细胞克隆数量均较0 nmol/L组减少（P＜0.05）。不同浓度的PF-4708671对vClub 向Club细胞的分化无明显影响,其特征分子Cyp2f2在mRNA表达水平差异方面无统计学意义。PF-4708671对Club 细胞向纤毛细胞和杯状细胞的分化能力均无明显影响,4组间2种细胞的标志物Act、Fox j1及Clca3、Foxa3表达水平 差异均无统计学意义（P＞0.05）。结论 mTOR/S6K1信号通路对小鼠气道干/祖细胞的增殖功能呈正调控作用,对其 分化功能影响甚微。     

Naltrexone and alcohol drinking in mice lacking beta-endorphin by site-directed mutagenesis

Alcohol-induced activation of the opioid system may contribute to the reinforcing properties of alcohol. This study investigated whether elimination of beta-endorphin (BE) synthesis via site-directed mutagenesis in embryonic stem cells would alter alcohol intake in mice. Both BE-deficient and wildtype (WT) mice generated from the targeted stem cells were backcrossed for nine generations onto a C57BL/6 background, and were maintained with ad libitum food and water. Mice had access to alcohol (10% v/v) under the following conditions: 24 h, scheduled access for 2 h/day, following acute (1 or 2 days) or chronic (5 weeks) alcohol deprivation, and scheduled access following six doses of naltrexone (0.125-16.0 mg/kg BW, ip) or saline treatment. Alcohol intake was similar in BE-deficient and WT mice given chronic access to alcohol, but greater in BE-deficient compared with WT mice during the first 10 days of scheduled access to alcohol, but not after more extensive experience with scheduled access. BE-deficient, but not WT mice, increased alcohol intake following 2 days, but not 1 day or 5 weeks, of deprivation. Naltrexone reduced alcohol drinking both in BE-deficient and WT mice, suggesting that drinking is mediated, in part, by activation of opioid receptors in both genotypes.     

Studies on the characteristics and mechanisms of testicular toxicity induced by Hydroxyurea

Abstract

Objective: Apoptosis plays a dominant role in both spontaneous spermatogenesis and germ cell death. This study was aimed to investigate the functions of related genes in testicular germ cell death induced by Hydroxyurea (HU).

Method: Wild-type (WT) and FasL transgenic (TG) DBA/C57BL mice were intraperitoneal injected with 400?mg/kg HU. Twelve hours later, testes were collected. Histomorphology of testis was observed by staining with Periodic Acid Schiff (PAS). Apoptosis was assessed by TUNEL assay. mRNA and protein levels of related genes were evaluated by quantitative RT-PCR and Western blot, respectively.

Results: The 2?×?2 factorial design comparative experiments between the WT and TG mice showed that the TG mice exhibited a higher basal apoptotic index. The basal mRNA levels of Fas and FasL and protein levels of Fas, FasL, Caspase-3, Caspase-8 and Caspase-9 in the TG mice were also higher than that in the WT mice. Twelve hours after injection of HU, the testicular tubules exhibited no significantly morphological changes but apoptosis index remarkably increased in both the WT and TG mice, with the latter having the higher amplitude. Although, HU up-regulated the mRNA of apoptosis-related genes, such as Fas and FasL, in both the TG and WT mice, the increased amplitude was more obvious in the TG mice. By Western blot analysis, apoptosis-related proteins Fas, FasL Caspase-3, Caspase-8 and Caspase-9 were significantly increased in both the WT and TG mice, with the TG mice exhibiting a greater up-regulation.

Conclusion: Germ cell apoptosis induced by the HU treatment may be related to the FasL-mediated signal transduction pathway.