Protein Expression of BLM Gene and Its Apoptosis Sensitivity in Hematopoietic Tumor Cell Strains

Patients with Bloom syndrome （BS） show an immunodeficiency, an enhanced sister chromatid exchanges （SCEs）, a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet （UV）- or hydroxyurea （HU）-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells expressed BLM protein higher than the normal human bone marrow mononuclear cells （P〈0.01）. In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV- or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the development of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic response.

O^6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY AND SENSITIVITY OF 20 CHINESE TUMOR CELL STRAINS TO 1-(4-AMINO-2-METHYL-5-PYRIMIDINYL) METHYL-3-(2-CHLOROETHYL)-3-NITROSOUREA

O6-methylguanine-DNA methyltransferase (MGMT) plays an important role in repairing alkylated DNA. MGMT activity as well as cellular sensitivity to 1- ( 4- amino- 2-methyl-5-pyrimidinyl) methyl-3- ( 2-chloroethyl)-3-nitrosourea (ACNU) of 20 Chinese tumor cell strains were assayed. A linear response between MGMT activity and ACNU sensitivity (D10) was observed. The lower the MGMT activity In the cells, the more the sensitivity to ACNU killing. It suggested that assay of MGMT activity in tumor biopsy could be used as a guide to predict the effectiveness of ACNU treatment in chemotherapy of human cancer.

Inhibitory effect of Fuzheng Yiliuyin in combination with chemotherapeutics on human gastric carcinoma cell strain

瞄准：在人的胃的癌房间紧张上与化学疗法在联合学习 fuzheng yiliuyin (为由加强身体抵抗压制肿瘤的煎) 的禁止的效果。方法：与化学疗法的各种各样的类型相结合的 Fuzheng yiliuyin (ZY ) 被放进二种有点胃的癌房间紧张，然后，它人的胃的癌房间紧张上的禁止的效果被 MTT 方法决定。流动血细胞计数器习惯于 apoptosis 评估的试金，并且胃的癌房间的超微结构在传播电子显微镜下面被观察。结果：明显的 apoptosis 为 72 h 与 ZY 在胃的癌房间术后疗法被看见。ZY 和化学的药在栽培的胃的癌房间上有协同的抑制效果，但是效果在各种各样的房间紧张上是不同的。ZY 的禁止的效果能被细胞毒素的行动和 apoptosis 加强。ZY 把化学疗法与氟尿嘧啶， etoposide 和 cisplatin (EFP ) 相结合最好 SGC-7901 上的禁止的效果，当 ZY 与 EFP 或与 DDP 化学疗法结合了时最好禁止的效果比 MGC-803 上的另外的药。结论：ZY 导致 apoptosis 并且禁止胃的癌细胞的生长。ZY 有化学疗法的协同的功能。

Expression of cyclooxygenase-2 mRNA in drug-sensitive cell and drug-resistant strains of ovarian cancer cell lines

Objective： To investigate the expression of cyclooxygenase-2 （COX-2） mRNA in drug-sensitive cell and drugresistant clones of ovarian cancer cell lines. Methods： RT-PCR and immunocytochemistry were used to investigate the expression of cyclooxygenase-2 in 3 clones drug-sensitive and 5 clones drug-resistant ovarian cancer cell. Results： Strong COX-2 mRNA expressions were detected in 3 clones of drug-sensitive cell and weak expressions were detected in 5 clones of drug-resistant cell. The protein expression of COX-2 in drug-sensitive cell was strongly positive reaction in immunocytochemistry stain and there was a weak positive reaction in 5 clones of drug-resistant cell. Conclusion： The expression of COX-2 mRNA in drug-sensitive cell strains is much higher than that in drugresistant strains of ovarian cancer cell lines, providing a basis of the chemoprevention for ovarian cancer.

GENOTOXIC EFFECT OF CIGARETTE SMOKE CONDENSATE (CSC) ON HUMAN DIPLOID CELL 2BS STRAIN

A series of bioassays such as sister chromatid exchange frequencies ( SCE.), chromosomal aberration ( CA ), micronuclel rate (MN) and cell-cycle delay have been used to detecting the genotoxic effect of cigarette smoke condensate (CSC) on human diploid cell 2BS strain. The results suggested that a higher SCE, ( 17. 0/ cell) was observed In 2BS cells treated with CSC at 100 μg/ml, as compared with 6. 9/cell of the background (P<0. 001). CA rate was significantly increased from 4% to 36% In cells treated with 10 μg/ml CSC (P< 0.001). MN rate varied from 9 -26‰ In cells treated with CSC compared to that of control (6‰). Meanwhile, the cell-cycle of cells was markedly delayed by CSC. The survival rate of 2BS cells declined to 59. 6% for treatment with CSC at 200 μg/ ml. There was a dose-effect response In SCE., CA, MN rate. We proposed that active oxygen might responsible for genotoxiclty of CSC on cells.

Sensing Traction Strain Induces Cell-Cell Distant Communications

Mechanobiology has been a highly recognized field in studying the importance of physical forces in physiologies at the molecular,cellular,tissue,organ and body-levels.Beside the intensive work focusing on the fine local biomechanical forces,the long-range force which can propagate through a relatively distant scale(in hundreds of micrometers and beyond)has been an intriguing topic with increasing attentions in recent years.The collective functions at cell population level often rely on cell-cell communications with or without direct contacts.Recent progresses including our own work indicate that the long-range biomechanical force propagating across scales far beyond single cell size may reserve the capability to trigger coordinative biological responses within cell population.Whether and how cells communicate mechanically in a distant manner remains largely to be explored.In respiratory system,the mechanical property of airway smooth muscle(ASM)is associated with asthma attack with prolonged contraction during airway hyper-responsiveness.In this work,we found that ASM cells rapidly self-assembled into a well-constructed network on 3D matrigel containing type I collagen(COL I),which required the collective functions and coordination of thousands of cells completed within 12-16 hours.Cells were assembled with aligned actin stress fibers and elongated nuclei.The assembling process relied on the long-range mechanical forces across the matrix to direct cell-cell distant interactions.We further found that single ASM cells could rapidly initiate multiple buds precisely pointing to neighboring cells in distance,which relied on cell traction force and force strain on the matrix.Beads tracking assay demonstrated the long-range transmission of cellular traction force to distant locations,and modeling of maximum strain distribution on matrix by finite element method predicted the consistency with cell directional protrusions and movements in experiments.Cells could sense each other in distance to move directionally on both non-fibrous matrigel and in much more efficient way when containing COL I.Cells recruited COL I from the hydrogel to build nearly identical COL I fibrous network to mechanically stabilize the cell network.Our results revealed that ASM cells can sense the traction strain transmitted through matrix to initiate distant communications and rapidly coordinate the network assembly at the population level through active cell-matrix interactions.As an interesting phenomenon,cells sound able to’make phone call’via the role of long-range mechanical force.In summary,this work demonstrated that long-range biomechanical force facilitates the collective functions of ASM cell population for network assembly.The cells reacted to traction strain on the matrix for distant communications,which resulted in directional budding and movement.Fibrous COL I had important roles in facilitating the efficiency of force transmission to induce the assembly and stabilizing the cell network.This work has helped advance the understanding of the feature andfunction of long-range biomechanical force at the cell population level.The observed high mechano-sensitivity of ASM cells might suggest a re-enforced feedback of enhanced contraction by excessive ASM under asthmatic condition.

ADVANCES IN THE STUDY ON LEUKEMIA STRAINS AND LEUKEMIA CELL LINES IN CHINA

The study and establishment of leukemia strains and leukemia cell lines have made great achievement in China.It has been extended from mice to rat model and related to explore human leukemia cell line and heterotransplantable tumor strains. The cellular types of leukemia strain have included Iymphocytic,tmyelocytic,ecythroleukmia and megakaryoblastic cell strain.

CD69+NK Cells Contribute to the Murine Hepatitis Virus Strain 3-Induced Murine Hepatitis

Summary： The role of hepatic CD69＋ natural killer （NK） cells in virus-induced severe liver injury and subsequent hepatic failure is not well defined. In this study, a mouse model of fulminant liver failure （FHF） induced by murine hepatitis virus strain 3 （MHV-3） was used to study the role of hepatic CD69＋NK cells in the development of FHF. The CD69 expression in NK cells in the liver, spleen, bone marrow and peripheral blood was detected by using flow cytometry. The correlation between the CD69 level in hepatic NK cells and liver injury was studied. The functional marker （CD107a）, and activating and inhibitory receptor （NKG2D and NKG2A） expressed on CD69＋NK cells and CD69-NK cells were detected by using flow cytometry. Pro-inflammatory cytokines （IL-9, IFN-y and TNF-a） were also examined by using intracellular staining. After MHV-3 infection, the number of CD69＋NK cells in the liver of BALB/cJ mice was increased markedly and peaked at 72 h post-infection. Similar changes were also observed in the spleen, bone marrow and peripheral blood. Meanwhile, the CD69 expression in hepatic NK cells was highly correlated with the serum level of ALT and AST. The expression of CD107a and NKG2D, as well as the production of TNF-a, IFN-7 and IL-9 in hepatic CD69＋NK cells was all significantly up-regulated during 48-72 h post-infection. In contrast, the NKG2A expression was increased in hepatic CD69-NK cells but not in CD69＋NK cells. These results suggested that hepatic CD69＋NK cells play a pivotal role in the pathogenesis of FHF by enhancing degranulation and cytotoxic ability of NK cells and increasing the production of pro-inflammatory cytokines.

Multiplication of the Recombinant Strain Re-7 of Avian Influenza Virus Subtype H5 in MDCK Cells

This study was conducted to explore the multiplication pattern of the recombinant strain Re-7 of avian influenza virus subtype H5 in Madin Darby Canine Kidney （MDCK） cells and to determine the optimal multiplicity of infection （MOI） and the optimal time for virus harvest. The recombinant strain Re-7 was inoculated at different MOIs into MDCK cells grown in serum-free medium in 100 L bioreactors for replication. Then, the hemagglutination（HA） titer, 50% tissue culture infectious dose （TCID50） and 50% embryo infectious dose （EID50） of culture medium were measured once every 12 h from 24 h after virus inoculation to determine the optimal MOI. After that, virus was inoculated at the optimal MOI determined above into MDCK cells for large-scale virus replication to determine the optimal time for virus harvest. The results showed that the optimal MOI was 10 2, and the optimal time for virus harvest was 60 h after inoculation. Under these conditions, the HA titer, TCIDso per 1 mL and EIDso per 0.1 mL were increased to 1：102 4, 10^7.33 and 10^6.83, respectively. This study provides relatively stable parameters for large-scale production of the recombinant strain Re-7 of avian influenza virus subtype H5.

高频超声在构建SD大鼠皮下移植性乳腺癌模型中的应用

目的:应用MADB-106大鼠乳腺癌细胞悬液皮下移植法建立SD大鼠乳腺癌移植性肿瘤动物模型,并使用高频超声监测肿瘤生长情况。方法:选取7~8周龄雌性SD乳鼠16只,将处于对数生长期的MADB-106大鼠乳腺癌细胞悬液于大鼠腹股沟皮下接种,应用高频超声观察成瘤率、肿块大小、血供情况及其它声像图特征。结果:SD大鼠接种成功率为100%,死亡2只,剩余14只生长良好;超声检查肿块为类圆形,内部低回声且稍不均匀,边界尚清,后方回声衰减,彩色多普勒扫查肿块内部及周边可见较丰富血流信号;3周后切除瘤体组织,大体观肿块成灰白色,多呈椭圆形,部分边缘呈分叶状改变,肿瘤血管较丰富,镜下可见肿瘤细胞大小形状各异,核大深染,异型性明显。结论:通过MADB-106大鼠乳腺癌细胞接种于雌性SD大鼠腹股沟皮下,成功建立了大鼠乳腺癌移植性肿瘤动物模型。高频超声技术可以在SD大鼠乳腺癌皮下成瘤过程中对肿物的大小、形态、边界、内部回声及血流等生物学情况进行动态检测,是观察和评价瘤体动态变化过程的重要技术手段。

Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.

天名精内生真菌的分离鉴定及抗肝癌活性研究

目的对天名精内生真菌进行分离和鉴定,并从中筛选出具有抗肝癌作用的活性菌株,为寻找新的抗肝癌先导化合物提供菌株资源。方法采用植物组织切块法和平板划线法对天名精内生真菌进行分离纯化;MTT法筛选对肝癌HepG2细胞增殖具有抑制作用的活性菌株,并计算活性显著内生真菌的IC50值;运用形态学鉴定和分子鉴定的方法对活性菌株进行鉴定。结果从天名精中共分离得到60株内生真菌,其中TMJ-03、TMJ-13、TMJ-15、TMJ-23、TMJ-54为活性内生真菌,其发酵物抑制HepG2细胞增殖的IC50值分别为19.49(TMJ-C-03)、31.13(TMJ-C-13)、88.65(TMJ-C-15)、37.42(TMJ-D-23)、17.22(TMJ-D-54)、32.72(TMJ-C-54)μg·mL^(-1)。5株活性菌株均鉴定到种,分别为Aspergillus terreus、Chaetomium globosum、Alternaria tillandsiae、Fusarium proliferatum、Alternaria arborescens。结论首次从天名精中筛选出对HepG2细胞增殖具有抑制作用的活性内生真菌,为寻找新的抗肝癌药物提供了菌株资源。

活菌制剂保护剂的作用机制与应用前景

活菌制剂因其独特的生物活性、稳定性以及便利性而被广泛应用于食品、化妆品、农业、环境治理以及医疗等多个领域。作者综述了菌种生理结构以及活菌制剂在生产加工及储藏过程中失活的影响因素,并分别列举了蛋白质、糖、脂质以及其他成分作为保护基质在活菌制剂中的作用机理,总结了不同属、不同生理状态的菌株对保护剂的不同需求,旨在为活菌制剂保护剂的开发和选择提供研究思路。

牛轮状病毒多克隆抗体制备及特性分析

旨在优化G6P[1]型牛轮状病毒(bovine rotavirus, BRV)在MARC-145细胞中的培养条件,制备其兔源多克隆抗体,并对制备的多抗进行效价检测及鉴定。对G6P[1]型BRV毒株NCDV进行不同病毒接种量及收毒时间优化,确定病毒在MARC-145细胞中的最佳培养条件;以1×10^(7) TCID_(50) NCDV活毒作为免疫原免疫成年兔,制备兔源多克隆抗体;通过间接ELISA检测多抗效价,并通过间接免疫荧光试验(IFA)及中和试验鉴定其特异性。结果:NCDV毒株以感染比0.1、感染时间24 h为最佳培养条件,病毒滴度可至4.9×10^(6) TCID_(50)/mL;间接ELISA结果显示,多克隆抗体的效价为1∶51 200,表明NCDV具有良好的免疫原性;IFA及中和试验结果表明,NCDV多克隆抗体与G6P[1]型、G9P[1]型和G10P[11]型BRV毒株均能发生特异性反应,存在良好的交叉中和效应,表明制备的多抗具有良好的反应性。综上,本试验成功优化了NCDV毒株在MARC-145细胞中的增殖条件,并制备了高效价的兔源多克隆抗体,为后续BRV感染的血清学诊断及疫苗研发奠定了理论基础。

Immortalized Rat Astrocyte Strain Genetically Modified by Rat Preprogalanin Gene

Summary: To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1(+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1(+)-GAL and pcDNA3.1(+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 μg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1(+)-GAL and pcDNA3.1(+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.

静态机械牵张力对HPDLSCs和PPDLSCs破骨相关基因表达的影响

目的:探讨健康牙周组织来源的牙周膜干细胞(HPDLSCs)和牙周病组织来源的牙周膜干细胞(PPDLSCs)在静态机械牵张力作用下破骨相关基因表达的异同。方法:采用低密度接种法分离培养HPDLSCs和PPDLSCs,应用流式细胞仪对两种细胞的表面间充质干细胞标记物表达进行检测,使用Flexcell Tension Unit对HPDLSCs和PPDLSCs进行不同力值静态机械牵张力(SMS)加载,实时定量RT-PCR检测HPDLSCs和PPDLSCs中破骨相关基因RANKL和C-fos的表达。结果:间充质干细胞表面标记物STRO-1、CD146、CD90、CD29在HPDLSCs和PPDLSCs中均强阳性表达,且HPDLSCs中的表达显著高于PPDLSCs(P<0.05)。在未加载SMS情况下,PPDLSCs中RANKL和C-fos的表达水平明显高于HPDLSCs(P<0.05);当加载SMS≤12%形变量时,HPDLSCs中两种破骨相关基因的表达水平较未加力时无明显变化(P>0.05),而形变量达到14%时,二者的表达显著上调(P<0.05);在PPDLSCs组,当SMS≤8%形变量时,RANKL和C-fos的表达无明显变化(P>0.05),而SMS≥10%形变量时,可明显激活两种破骨基因的表达(P<0.05)。结论:HPDLSCs和PPDLSCs对SMS的反应不同,过大的静态牵张力会导致PPDLSCs表达破骨相关基因增强。

多维约束称重提高皮带秤测量精度研究应用

通过分析多维约束称重传感器特征、整体结构和称重精度的影响因素、校准方式等,提出采用全桥连接方式连接电阻应变片,以获得较好的输出特性。实际应用表明,动态测量累计误差值低于0.5%,解决了皮带秤测量精度较低问题。

Providencia rettgeri strain HNR菌株羟胺氧化酶超声波法提取的研究

为了更有效和便捷的提取菌株脱氮过程中的关键酶,本文利用超声波破碎法对异养脱氮菌Providencia rettgeri strain HNR进行破碎,通过单因素实验及正交试验,确定出最佳破碎条件,便于后续试验。实验结果表明:最佳超声破碎条件为菌液OD_(600)为1.996,工作次数为70次,工作/间歇为5/5s功率为400W的组合为最佳工作条件。在此工作条件下破碎后得到的酶粗提液对羟胺有降解,得到羟胺氧化酶。

浅层膨胀土及其纤维改良土的剪切强度特性

与膨胀土工程灾害密切相关的浅层膨胀土受到的围压通常较低,其力学特性有别于高围压下的情形.通过三轴剪切试验比较高、低围压下膨胀土的剪切行为及剪切强度的非线性特征,探讨聚丙烯纤维改良浅层膨胀土的剪切特性及加固机理.结果表明,浅层膨胀土剪切强度具有明显的非线性,可用幂函数表征.随着土体中水的质量分数增大和围压减小,膨胀土剪切强度非线性更加明显.采用高围压下的抗剪强度参数试验结果会导致浅层饱和膨胀土的黏聚力被高估225.7%、内摩擦角被低估42.5%.掺入纤维后膨胀土的剪切强度明显提高,提升幅度与土体中的三维纤维网状结构的形成度有关.增大纤维长度可以有效降低剪切强度衰减率,继而消弱膨胀土的应变软化效应;过长的纤维在膨胀土内容易弯曲和扭折,会导致纤维-膨胀土界面作用随着土体变形而逐渐劣化.

Dauricine can inhibit the activity of proliferation of urinary tract tumor cells

Objective:To explore the anti-tumor effects of asiatic moonseed rhizome extraction-dauricine on bladder cancer EJ cell strain,prostate cancer PC-3Mcell strain and primary cell culture system.Methods:The main effective component-phenolic alkaloids of Menispermum dauricum was extracted and separated from asiatic moonseed rhizome by chemical method.MTT method was used to detect dauricine anti-tumor effect.Results:Dauricine had an obvious proliferation inhibition effect on the main tumor cells in urinary system.The minimum drug sensitivity concentration was between 3.81-S.ISμg/mL,and the inhibition ratio increased with the increase of concentration.Condusioiis:Dauricine,the main effective component extracted from asiatic moonseed rhizome,had a good inhibition effect on tumor cells in urinary system.At the same time,Dauricine has certain inhibition effects on the primary cultured tumor cell.