ESTABLISHMENT OF A MURINE ASCITES HEPATOMA CELL LINE H_(22)-F_(25)/L AND ITS BIOLOGICAL CHARACTERISTICS

Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak tune of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was Increased by 25 tunes. H22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo)was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H22- F25/ L Is a population of heterogenetlc tumor cells Including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.

Inhibitory effect of parvovirus H-1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability in vitro of QGY-7703, a cultured human hepatoma cell line, and on the formation and growth of its tumors in nude mice was studied. With higher multiplicity of infection (MOI) of H-1 given, survival of the QGY-7703 cells was found to be decreased. H-1 DNA amplification level at 30 h postinfection(p.i.) was detected to be 7.4 times higher than that at 2 h by dispersed cells assay, while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells (new-born human kidney cell line, NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay. The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2 h postinfection was observed to be prevented in 2 groups with given MOI 25 and 50. The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20 d latent period. It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.

Study on regulating mechanisms of oxocrebanine obtained from Stephania hainanensis H.S.Lo et Y.Tsoong on microtubule sites and tubulin in human breast cancer MCF-7 cells

Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.

Effects of PI3K Inhibitor NVP-BKM120 on Acquired Resistance to Gefitinib of Human Lung Adenocarcinoma H1975 Cells

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~unol/L）, and NVP-BKM120 （1 ~tmol/L） or NVP-BKM120 （1 pmol/L） plus gefitinib （1 ~tmol/L） obviously inhibited the activation of Akt, $6 and 4E-BP1 as compared with control group, but single use of gefitinib （1 pmol/L） exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H 1975 cells to gefitinib.

医用重组人源胶原蛋白功能敷料体外免疫原性评价

目的对一种医用重组人源胶原蛋白功能敷料的体外免疫原性进行评价。方法以细胞培养液为浸提介质,按0.2 g/ml的比例浸提制备试验液,进行体外淋巴细胞增殖试验、人细胞系激活试验(h-CLAT)、细胞炎症因子含量检测。结果体外淋巴细胞增殖试验中,100%,50%,25%3种浓度试验液的淋巴细胞增殖率分别为105%,111%,86.3%,与阴性对照组比较,差异无统计学意义(P>0.05)。h-CLAT结果显示,试验组THP-1细胞表面分子CD54 RFI值<200,CD86 RFI值<150,判定为阴性结果。试验组细胞上清中肿瘤坏死因子α(TNF-α)、白介素8(IL-8)的含量与阴性对照组比较,差异无统计学意义(P>0.05)。结论医用重组人源胶原蛋白功能敷料有良好的生物相容性。

几种壳聚糖类产品的致敏试验研究

目的通过豚鼠最大剂量试验(GPMT)、小鼠局部淋巴结试验(LLNA)、人细胞系激发试验(h-CLAT)探究几种壳聚糖类产品的致敏性。方法将羧甲基壳聚糖、壳聚糖止血粉、羧甲基壳聚糖愈创膏、羧甲基壳聚糖止血纱作为试验样品,按GB/T 16886.12规定的比例浸提制备受试液,按GB/T 16886.10-2017进行GPMT、LLNA试验,按OECD442(E)进行h-CLAT试验,对样品进行致敏性检测。结果羧甲基壳聚糖、壳聚糖止血粉、羧甲基壳聚糖愈创膏、羧甲基壳聚糖止血纱的GPMT、LLNA试验结果均为阴性。h-CLAT试验中,羧甲基壳聚糖、羧甲基壳聚糖愈创膏、羧甲基壳聚糖止血纱的综合判定结果为阴性;壳聚糖止血粉100%、50%、25%浓度受试液CD54的相对荧光强度(RFI)均高于2.00,CD86的RFI均高于1.50,综合判定结果为阳性。结论采用作为皮肤致敏评价体外替代方法之一的h-CLAT评估壳聚糖止血粉的致敏反应风险有待于进一步研究。

Influence of Toxoplasma gondii on in vitro proliferation and apoptosis of hepatoma carcinoma H7402 cell

Objective:To discuss the influence of tachyzoite of Toxoplasma gondii（T.gondii） RH strain on proliferation and apoptosis of hepatoma carcinoma（HCC） H7402 cell.Methods:The HCC H7402 cell in logarithmic phase and tachyzoite of T.gondii RH strain in different concentrations（1×107/mL,2×107/mL.4×107/mL,8×107mL and 16×107/mL） were co-cultured.CCK-8was utilized to determine the inhibition rate of T.gondii tachyzoite on H7402 cell growth.Flow cytometry was used to detect the change of cell cycle.RT-PCR method was used to detect the expression of cyclinB1 and cdc2-two genes related to cell cycle.Western blot method was used to detect the expression of apoptosis-related proteins Caspase-3 and Bcl-2.Results:The tachyzoite of T.gondii RH strain can inhibit the proliferation of HCC H7402 cells.The inhibition rate of tumor cell growth increased with the increase of concentration of T.gondii tachyzoite.With the increase of concentration of T.gondii tachyzoite,the proportion of G0/G1 phase of H7402 cell increased,the proportion of S phase decreased,and PI value decreased accordingly.The expression of cyclinB1 and cdc2 genes decreased with the increase of the concentration of T.gondii tachyzoite.With the increase of the concentration of tachyzoite of T.gondii RH strain,the expression quantity of Caspase-3 in H7402 cell increased,but the expression quantity of Bcl-2protein decreased.Conclusions:T.gondii can inhibit the in vitro proliferation of HCC H7402 cell,and induce its apoptosis.This effect shows a trend of concentration-dependent increase.Moreover,it is related to the down-regulation of cyclinB1 and cdc2（cell cycle-related genes）,the increase of apoptosis-related protein Caspase-3.and the decreasc of Bcl-2 expression.

青蒿琥酯抗肝癌作用的实验研究

目的 :研究青蒿琥酯的抗肝癌作用。方法 :运用小鼠肝癌H2 2 癌模型、MTT法和集落形成法 ,观察青蒿琥酯对荷瘤鼠和人肝癌SMMC 772 1细胞株的作用。结果 :口服青蒿琥酯 30 0mg·kg-1·d-1,肿瘤受到明显抑制 ,抑瘤率在 3次实验中分别为 49.1% ,48.7% ,46 .6 % ;小鼠生存时间明显延长。青蒿琥酯与 5 氟尿嘧啶有协同抗肿瘤作用。青蒿琥酯体外对人肝癌细胞有明显细胞毒作用 ,其IC50 为 2 .0 7μg·ml-1,同时抑制人肝癌SMMC 772 1克隆原细胞形成集落 ,其IC50 为 2 .48μg·ml-1。结论

消瘤平移合剂含药血清诱导肝癌H-7402细胞凋亡的实验研究

目的 :观察消瘤平移合剂含药血清对肝癌H 74 0 2细胞凋亡的诱导作用。方法 :运用血清药理学方法 ,选择不同浓度的含药血清与人肝癌H 74 0 2细胞共同孵育 ,于不同时间观察 ,并与空白血清作对照。结果 :消瘤平移合剂含药血清可显著抑制H 74 0 2细胞的增长 ;H 74 0 2细胞在显微镜下呈现典型的凋亡形态学改变 ,流式细胞仪检测可见亚G1峰 ,凋亡呈含药血清浓度、时间依赖性特点 ,对照组及 5 %、10 %含药血清作用 2 4h ,其凋亡率分别为 0 4 9%、5 5 8%、8 4 6% ,4 8h凋亡率分别为 0 83%、11 5 8%、14 2 5 % ;bcl 2基因检测显示bcl 2基因表达受抑。结论 :消瘤平移合剂可通过诱导肿瘤细胞凋亡 ,发挥其临床抗肿瘤、抗转移的作用。

人白血病细胞裸鼠异种移植模型的建立

我们成功地将人Ｔ淋巴细胞白血病细胞、人早幼粒细胞白血病细胞移植到裸鼠体内。裸鼠经环磷酰胺预处理后分别将（５－１０）×１０＾６ＭＯＬＴ－４细胞及（２－４）×１０＾６ＨＬ－６０细胞移植到动物皮下能形成实体瘤，该肿瘤呈进行性生长。

DHA复合物对鼠移植瘤细胞和T淋巴细胞细胞周期及凋亡的影响

目的 研究DHA复合物的抗癌机理。方法 用流式细胞术研究了DHA复合物对荷瘤鼠H2 2 细胞和T淋巴细胞细胞周期及凋亡的影响。结果 ( 1)与阴性对照组相比 ,DHA复合物中、高剂量组G0 G1期H2 2 癌细胞百分比明显增加 (P <0 .0 1) ;DHA复合物低、中、高剂量组G0 G1期T细胞百分比明显减小 (P <0 .0 1)。 ( 2 )与阴性对照组相比 ,DHA复合物低、中、高剂量组S期H2 2 癌细胞百分比明显减小 (P <0 .0 1) ,S期T细胞百分比明显增加 (P <0 .0 1)。 ( 3)与阴性对照组相比 ,DHA复合物中剂量组G2 M期H2 2 癌细胞百分比显著减少 (P <0 .0 1) ,DHA复合物低、高剂量组G2 M期H2 2 癌细胞以及DHA复合物低、中、高剂量组G2 M期T细胞百分比均显著升高 (P <0 .0 1)。 ( 4 )与阴性对照组相比 ,DHA复合物低、中、高剂量组H2 2 癌细胞增殖指数 (PI)明显下降 (P <0 .0 1) ,T细胞增殖指数明显升高 (P <0 .0 1)。 ( 5 )与阴性对照组相比 ,DHA复合物低、中、高剂量组H2 2 癌细胞及DHA复合物中、高剂量组T细胞凋亡率明显升高 (P <0 .0 1)。结论 DHA复合物可抑制H2 2 癌细胞的增殖 ,促进T淋巴细胞增殖 ,同时DHA复合物还可促进H2 2 细胞。

小鼠LAK细胞MHC表达水平对其体外杀伤肿瘤活性的影响

目的探讨LAK细胞表面MHC的表达水平与体外肿瘤细胞杀伤活性的关系。方法使用siRNA沉默小鼠LAK细胞MHC-Ⅰ(H-2Kd)分子的表达,同时设空转染组和无关序列组,流式细胞仪检测不同组别靶蛋白的表达,LDH释放试验检测H-2Kd表达降低的LAK细胞对K562和H22肿瘤细胞株的杀伤活性。结果流式细胞检测证实siRNA能够抑制靶蛋白的表达,H-2Kd表达降低组对K562和H22肿瘤细胞的杀伤活性与对照组相比明显降低,空转染组和无关序列组与对照组无显著性差异。结论小鼠脾LAK细胞表面H-2Kd的表达水平与其体外杀伤活性相关。

抗肿瘤药物与TRAIL联用对喉癌HEP-2细胞株增殖、凋亡的影响

目的探讨化疗药物顺铂(DDP)、5氟尿嘧啶(5-Fu)单独及与TRAIL联合对HEP-2细胞株增殖、凋亡的影响。方法MTT法检测其增殖抑制作用,FCM法检测细胞凋亡率。结果TRAIL与5-Fu、DDP联合作用于HEP-2细胞株时,其抑制率、凋亡率高于单独作用时的抑制率和凋亡率。结论TRAIL与5-Fu、DDP联合作用有协同抑制HEP-2细胞株增殖、诱导HEP-2细胞株凋亡的作用。

mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响

目的探究mCD99L2基因沉默对小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞的影响。方法重组SiRNA表达质粒LV-mCD99L2,体外转染内源性mCD99L2表达阳性的A20细胞,筛选出稳定表达LV质粒的细胞株并扩增培养;采用免疫荧光技术和流式细胞仪检测转化前后两组细胞鼠源CD30表达;透射电镜观察转化后细胞超微结构的形态特点;细胞计数方法动态观测培养细胞干扰组A20-LV-mCD99L2和未经干扰组A20细胞的H/RS样细胞(直径≥25μm)转型率,以人霍奇金淋巴瘤细胞系L428作为对照;采用流式细胞仪检测细胞周期变化。结果获得了稳定表达LV质粒的单克隆细胞株A20-LV-mCD99L2;免疫荧光标记显示转化细胞CD30(+);流式细胞仪检测A20-LV-mCD99L2细胞CD30阳性率为54.4%;透射电镜观察转化后细胞核增大,可见单核、双核及多核,核仁明显的H/RS样细胞;干扰组H/RS样细胞的转型率明显高于未经干扰组(P<0.01)。两组处于S期的细胞无明显差异,两组细胞均未见凋亡峰。结论mCD99L2基因沉默可诱导小鼠B淋巴瘤细胞系A20细胞转化为H/RS样细胞。

nm23-H_1基因缺失人肺癌细胞株的筛选与鉴定

nm23-H1基因与肺癌的侵袭与转移密切相关,但是其作用的分子机制尚不清楚,为研究nm23-H1基因的功能,筛选并鉴定了nm23-H1基因缺失人肺癌细胞株及其生物学特性.应用Southernblot,RT-PCR和West-ernblot检测9株人肺癌细胞株中nm23-H1基因的存在状态及其生物学行为.结果发现发现人大肺癌细胞株L9981中存在nm23-H1等位基因的杂合性缺失,与其同源的NL9980及其它7株肺癌细胞株中nm23-H1基因均以杂合子的形式存在;并且L9981细胞株的增殖能力、克隆形成能力、体外侵袭力,裸鼠体内成瘤性及移植瘤肺转移的能力均显著高于NL9980.研究结果显示nm23-H1基因的缺失可能与L9981细胞株恶性表型和高转移潜能密切相关.

几种药用真菌粗提物对多种人体肿瘤细胞株增殖的抑制作用

本项研究直接采用各种人体肿瘤细胞株作为供试体 ,检测了几种药用真菌粗提物在体外对不同人体肿瘤细胞增殖的抑制作用 ,建立了一套筛选抗肿瘤药物的体外检测系统。检测样品对肿瘤细胞增殖的抑制作用采用比利时 Biosource公司的 AlamarBlue Assay试剂盒。本项研究检测了云芝、灵芝、猴头和灰树花四种药用真菌的粗提物( YZ、LZ、HT和 HSH)对五种人体肿瘤细胞株 :慢性骨髓性白血病细胞株 ( K562 )、大肠腺癌细胞株 ( SW62 0 )、乳腺腺癌细胞株 ( MCF7)、急性 T淋巴白血病细胞株 ( Jurkat)和大肠腺癌细胞株 ( L S180 )增殖的抑制作用。结果显示 ,四种药用真菌粗提物对 K562、SW62 0和 Jurkat的增殖都有不同程度的抑制作用。相比之下 ,YZ对 K562和 SW62 0的抑制作用最强。而 HT对 Jurkat的抑制作用最强 ,只有 YZ和 L Z对 L S180的增殖有明显的抑制作用。L Z在高浓度下不仅对 K562、SW62 0和 Jurkat,而且对 L S180的增殖抑制作用显著。四种药用真菌粗提物对 MCF7都没有明显的抑制作用。本项研究表明 ,不同的药用真菌粗提物对不同的肿瘤细胞株的增殖有不同的抑制作用 ,显示出抗肿瘤真菌具有一定的专效性。这对有目的地进行新的抗肿瘤药物的筛选和进一步在分子水平上研究抗肿瘤真菌的药理具有重要的意义。

直接多肽结合试验组合人细胞系活化试验预测皮肤致敏物的探讨

目的建立直接多肽结合试验(DPRA)和人细胞系活化试验(h-CLAT)的皮肤致敏组合检测方法,对化学品及植物提取物的致敏性进行初筛。方法选择12种化学品和7种植物提取物为受试物,把不同受试物分别与两种肽(半胱氨酸肽和赖氨酸肽)共孵育24 h,采用高效液相色谱法分析反应后多肽消耗。同时将不同浓度的受试物与体外培养的人急性单核细胞白血病细胞(THP-1)共孵育24 h,通过流式细胞仪检测暴露后细胞表面标志物CD86和CD54的变化。再进一步比较DPRA和h-CLAT预测结果的一致性。结果 DPRA方法和h-CLAT方法准确区分了12种化学品的皮肤致敏性,其中2种判定为阴性,10种判定为阳性,两种方法检测结果一致。DPRA对植物提取物致敏性的预测中,除绿茶提取物无法判定外,其余6种均为皮肤致敏疑似物质。h-CLAT预测中,除绿茶提取物、马齿笕提取物和人参果提取物为非致敏物,其余4种植物提取物为致敏物。DPRA与h-CLAT预测的一致性为0.57。结论 DPRA与h-CLAT的简单组合可以实现对单一化合物的准确预测,对于复杂混合物可实现初步预测,确认需要进一步组合其他方法。

基于THP-1细胞的皮肤致敏体外检测方法

目的建立基于人细胞系的替代皮肤致敏动物实验的体外检测方法(h-CLAT),并对化学品、日用化学产品和化妆品植物原料进行皮肤致敏性检测。方法体外培养人急性单核细胞白血病细胞(THP-1),不同浓度受试物与细胞共孵育24 h,通过流式细胞仪检测细胞表面标志物CD86和CD54活化的差异,并对11种已知皮肤致敏化学物质和9种未知样品进行皮肤致敏性预测。同时对未知样品进行豚鼠局部封闭涂皮法验证。结果使用h-CLAT方法准确区分了11种参考物质的皮肤致敏性,9种受试样品中,7种样品被判断为阴性皮肤致敏物质,2种植物提取物被确认为皮肤致敏疑似物质。9种样品的预测结果与动物实验一致。结论 h-CLAT体外检测方法可以代替部分动物测试,用于可溶性皮肤致敏物质的预测。

小分子化合物H-131抑制胃癌细胞增殖及机制

目的研究小分子化合物H-131对人胃癌细胞SGC7901、BGC823、MKN-45增殖的抑制作用及其相关机制。方法应用不同浓度的化合物H-131处理人胃癌细胞SGC7901、BGC823和MKN-45,采用MTT法检测该化合物对细胞增殖的影响,流式细胞术检测其对细胞周期的影响,Western blot检测化合物对周期调控蛋白Cyclin D1和CDK4表达的影响。结果化合物H-131能够以剂量依赖的方式抑制人胃癌细胞SGC7901、BGC823及MKN-45的增殖,并且能够抑制上述细胞G1期向S期的转化。Western blot结果显示H-131能够以剂量依赖的方式下调胃癌细胞SGC7901中Cyclin D1和CDK4的蛋白表达水平。结论化合物H-131能够通过下调胃癌细胞中Cyclin D1和CDK4的表达水平,抑制胃癌细胞G1期向S期的转化,进而抑制胃癌细胞增殖。