血管生成抑制剂-细胞毒药物联用对胰腺癌生长和转移的实验治疗

目的 探讨血管生成抑制剂 细胞毒药物联用疗法 [TNP 4 70 / 2′ ,2′ 二氟脱氧胞苷 (Gemc itabine) ]对胰腺癌裸鼠原位移植模型 (SOI)生长、转移的抑制作用。方法 单用疗法 :2 4只胰腺癌SOI模型随机分为T3 0组 (TNP 4 70 3 0mg/kg ,皮下注射 ,隔日一次 ,疗程 8周 )、G10 0组 (Gemcitabine 10 0mg/kg ,腹腔内给药 ,术后第 0、3、6、9天 )和对照组 ;联用疗法 :3 2只SOI模型随机分为T15组、G5 0组、对照组和联用组 (采用T3 0 +G5 0 ) ,术后第 10周处死裸鼠。结果 G10 0侧重于抑制胰腺癌生长 ,T3 0则侧重抑制胰腺癌转移 ,两者均无显著的改善预后作用 ;T15和G5 0无显著的抗肿瘤生长和转移作用 ;只有联用组能显著抑制胰腺癌生长、转移和改善预后 ,T3 0可使G5 0的抑瘤率提高 2倍 ,并获得 2 / 8只的肿瘤治愈率 ;T3 0组的微血管密度为 7.13± 2 .99/高倍视野 ,显著低于T15组的 10 .5 3± 3 .2 2 /高倍视野和对照组的 14 .5 0± 5 .93 /高倍视野 (P <0 .0 5 )。结论 血管生成抑制剂 细胞毒药物联用疗法 (TNP 4 70 3 0mg/kg和Gemcitabine 5 0mg/kg)能有效抑制肿瘤生长和转移 ,有助于减少细胞毒药物剂量及不良反应 ,为安全有效的抗肿瘤策略。

骨髓基质干细胞(BMSCs)细胞毒最佳实验条件的研究

目的探讨CCK-8法和MTT法在骨髓基质干细胞(Bone marrow stromal cells,BMSCs)细胞毒实验中的最佳实验条件.方法取对数生长期的P3大鼠BMSCs,制成不同浓度的细胞悬液,于96孔培养板中培养24 h,分别用CCK-8法、MTT法检测其吸光度(A)值,根据细胞浓度与A值关系曲线确定两种方法各自的最适细胞浓度;取最适浓度的细胞于0.5,1,2,3,4和5 h 6个不同时间点,分别于450,490,595 nm处测定吸光度A值,确定两种方法最佳的检测时间和最适检测波长;采用CCK-8和MTT法检测5个浓度的鹿茸Ⅰ型胶原对BMSCs的增殖的影响.结果 CCK-8法测得的A值与细胞数目之间的线性相关性优于MTT法;CCK-8法、MTT法最佳检测波长分别为450,490 nm,最佳检测时间分别为1,4 h;在检测鹿茸Ⅰ型胶原蛋白对BMSCs的增殖的影响时,CCK-8法测得数据的线性相关性优于MTT法,并且数据偏差较MTT法小.结论 CCK-8法与MTT法检测结果相比线性相关性好,灵敏度高,准确性好.在BMSCs细胞毒检测中CCK-8法优于MTT法.

改变病情的抗风湿药物(一)

改变病情的药物（disease modifying antirheumatic drugs，DMARDs）又称慢作用药、细胞毒药，对延缓风湿病的关节破坏有一定作用，是目前治疗风湿病的主要药物。临床常用的制剂包括甲氨喋呤、环磷酰胺、硫唑嘌呤、来氟米特、羟氯喹、吗替麦考酚酯、环孢霉素、他克莫司、柳氮磺胺吡啶、青霉胺以及金制剂。

编者的话

在现代药物治疗方面，从第一个化疗药物氮芥的问世至今已有半个多世纪了，很多新的化疗药物，在肿瘤的治疗中取得了很好的疗效。然而化疗药物普遍具有针对性不强、不良反应较大的缺点。近年来，我们终于迎来了分子靶向药物的新时代。与以往的细胞毒药物相比，分子靶向药物针对性更强、毒性更低，

以消化系统受累为首发症状的原发性小血管炎2例

例1男，56yr。因剧烈上腹疼痛、伴恶心、呕吐8h来院就诊。查体：T38．5℃，BP120／75mmHg，上腹压痛、反跤痛(+)。心电图排除心肌梗死，腹透未见液气平。血常规：WBC16．7×10^9·L^-1，HGB86g·L^-1。血沉86mm·h^-1。腹部B超显示：胰腺体积增大。血淀粉酶1800U。拟诊断“急性胰腺炎”收人院。按急性胰腺炎处理。

Reversal of Multidrug Resistance by Neferine in Adriamycin Resistant Human Breast Cancer Cell Line MCF-7/ADM

Objective: To study the reversal effect of neferine on adriamycin (ADM) resistant human breast cancer cell line MCF-7/ADM. Methods: The cytotoxic effect of Nef or ADM was determined by 3-[4, 5-dimethylthiazol-2.-yl], 5-diphenyl tetraxolium bromid (MTT) assay. Apoptosis and the expression of P-glycoprotein (P-gp) were detected by flow cytometry (FCM). The intracellular ADM concentration was measured by HPLC. Results: Nef at 1, 5, 10 mol/L decreased the IC50 of ADM to MCF-7/ADM from 11.63 g/mL to 4.59, 2.44, 0.27 g/mL respectively. MCF-7/ADM could resist the apoptosis induced by ADM while Nef (1-10 mol/L) could augment ADR-mediated apoptosis. Nef (10 mol/L) increased the accumulation of ADM up to 2.88 fold in MCF-7/ADM but not in sensitive cells MCF-7/S and reduced the expression of P-gp in MCF-7/ADM cells. Conclusion: Nef can circumvent multidrug resistance (MDR) of MCF-7/ADM cells and the mechanism was associated with the increase of intracellular accumulation of ADM and the reduced expression of P-gp in MCF-7/ADM cells.

Developments in metastatic pancreatic cancer:Is gemcitabine still the standard?

In the past 15 years, we have seen few therapeutic advances for patients with pancreatic cancer, which is the fourth leading cause of cancer-related death in the United States. Currently, only about 6% of patients with advanced disease respond to standard gemcitabine therapy, and median survival is only about 6 mo. Moreover, phase Ⅲ trials have shown that adding various cytotoxic and targeted chemotherapeutic agents to gemcitabine has failed to improve overall survival, except in cases in which gemcitabine combined with erlotinib show minimal survival benefi t. Several metaanalyses have shown that the combination of gemcitabine with either a platinum analog or capecitabine may lead to clinically relevant survival prolongation, especially for patients with good performance status. Meanwhile, many studies have focused on the pharmacokinetic modulation of gemcitabine by fi xed-dose administration, and metabolic or transport enzymes related to the response and toxicity of gemcitabine. Strikingly, a phase Ⅲ trial in 2010 showed that, in comparison to gemcitabine alone, the FOLFIRINOX regimen in patients with advanced disease and good performance status, produced better median overall survival, median progression-free survival, and objective response rates. This regimen also resulted in greater, albeit manageable toxicity.

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC)vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d).METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization,the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d.The survival rate and living time of mice were also calculated.RESULTS: After 4 wk of DC immunization, the A450 nm values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56±0.17 and 0.12±0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were(73.2±3.1) % and (24.4±8.8) %, which were significantly higher than those of mice injected with water.The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered.CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc.Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Effects of a 24-week course of interferon-αtherapy after curative treatment of hepatitis C virus-associated hepatocellular carcinoma

AIM： To assess whether a 24-wk course of interferon （IFN） could prevent hepatocellular carcinoma （HCC） recurrence and worsening of liver function in patients with hepatitis C virus （HCV）-infected patients after receiving curative treatment for primary HCC.METHODS： Outcomes in 42 patients with HCV infection treated with IFN-α, after curative treatment for primary HCC （IFN group）, were compared with 42 matched curatively treated historical controls not given IFN （non- IFN group）.RESULTS： Although the rate of initial recurrence did not differ significantly between ]FN group and non-IFN group （0%, 44%, 61~, and 67% ys 4.8%, 53~, 81%, and 87% at 1, 3, 5, and 7 years, P = 0.153, respectively）, ]FN group showed a lower rate than the non-IFN group for second recurrence （0%, 10.4%, 28%, and 350/0 ys 0%, 30~ , 59%, and 66% at 1, 3, 5 and 7 years, P = 0.022, respectively）. Among the ]FN group, patients with sustained virologic response （SVR） were less likely to have a second HCC recurrence than ]FN patients without an SVR, or non-IFN patients. Multivariate analysis identified the lack of SVR as the only independent risk factor for a second recurrence, while SVR and Child-Pugh class A independently favored overall survival.CONCLUSION： Most intrahepatic recurrences of HCV-related HCC occurred during persistent viral infection. Eradication of HCV is essential for the prevention of HCC recurrence and improvement of survival.

Inhibition effect of Chinese herbal medicine on transcription of hepatitis C virus structural gene in vitro

AIM: To investigate the inhibitory effect of Chinese herbal medicine on the transcription of hepatitis C virus (HCV) structural gene in Hela D cells.METHODS: Hela cell line was transfected with recombinant pBK-CMV-HCV containing HCV structural gene by Lipofectamine. RT-nested-PCR and Western blot assay were used to testify the HCV gene expression in Hela cells. The Hela cells expressing HCV structural protein were named Hela D cells. Prescriptions of Xiao chaihu Decoction (XCHD),Fufang Huangqi (FFHQ) and Bingganling (BGL) wererespectively added to Hela D cells in various concentrations. Semi-quantitative RT-nested-PCR product analysis was performed according to the fluorescent density between HCV DNA band and GAPDH DNA band in gel electrophoresisafter screened. RESULTS: Recombinant pBK-CMV-HCV could correctly express the HCV structural gene in Hela D cells. After coculture of Hela D cells with three prescriptional different concentrations for 48 h respectively, the transcription of HCVgene decreased with increasing of the concentration of each prescription. The lightness ratio of HCV product bands to GAPDH product bands was 0.24, 0.10 and 0.12 in Hela D cells incubated with 0.1 g/mL of XCHD, FFHQand BGL respectively and the lightness ratio HCV product bands to GAPDH product bands was 0.75, 0.67 and 0.61respectively in the control cells. CONCLUSION: The prescriptions of XCHD, FFHQ and BGL partly inhibit the transcription of HCV structural gene inHela D cells.

Effect of JIANPI HUOXUE decoction on inflammatory cytokine secretion pathway in rat liver with lipopolysaccharide challenge

AIM： To evaluate the effect of Chinese traditional medicinal prescription, JIANPI HUOXUE decoction （JHD） on cytokine secretion pathway in rat liver induced by lipopolysaccharide （LPS）. METHODS： Twenty-four male SD rats were divided into normal group （n = 4）, model group （n = 10） and JHD group （n = 10） randomly. Rats in model group and JHD group were administrated with normal saline or JHD via gastrogavage respectively twice a day for 3 d. One hour after the last administration, rats were injected with LPS via tail vein, 50 μg/kg. Simultaneously, rats in normal group were injected with equivalent normal saline. After LPS stimulation for 1.5 h, serum and liver tissue were collected. Pathological change of liver tissues was observed through hematoxylineosin （H.E.） staining. Tumor necrosis factor alpha （TNF-α） in serum were assayed by enzyme linked immunosorbent assay （ELISA）. The protein expression of TNF-α, phosphorylated inhibit-κB （p-κB） and CD68 in liver were assayed by Western blot. The distribution of CD68 protein in liver was observed through immunohistochemical staining. The mRNA expression of TNF-α, interleukin-6 （IL-6）, CD14, toll-like receptor 2 （TLR2） and TLR4 in liver were assayed by real-time RT-PCR.RESULTS： Predominant microvesicular change, hepatocyte tumefaction and cytoplasm dilution were observed in liver tissues after LPS administration as well as obvious CD68 positive staining in hepatic sinusoidal. After LPS stimulation, serum TNF-α （31.35 ± 6.06 vs 12225.40 ± 9007.03, P 〈 0.05）, protein expression of CD68 （1.13 ± 0.49 vs 3.36 ±1.69, P 〈 0.05）, p-IκB （0.01 ±0.01 vs 2.07 ＋ 0.83, P 〈 0.01） and TNF-α （0.27 ± 0.13 vs 1.29 ± 0.37, P 〈 0.01） in liver and mRNA expression of TNF-α （1.96 ± 2.23 vs 21.45 ±6.00, P 〈 0.01）, IL-6 （4.80 ± 6.42 vs 193.50 ± 36.36, P 〈 0.01） and TLR2 （1.44 ± 0.62 vs 4.16 ± 0.08, P 〈 0.01） in liver were also increased significantly. These pathological changes were all improved in .1HD group. On the other hand, TLR4 mRNA （1.22 ± 0.30 vs 0.50 ± 0.15, P 〈 0.05） was down-regulated and CD14 mRNA increased but not significantly after LPS stimulation. CONCLUSION： JHD can inhibit cytokine secretion pathway induced by LPS in rat liver, which is probably associated with its regulation on CD68, p-IκB and endotoxin receptor TLR2.

Ca^(2+) cytochemical changes of hepatotoxicity caused by halothane and sevoflurane in enzyme-induced hypoxic rats

AIM： To investigat the relation between hepatotoxicity of halothane and sevoflurane and altered hepatic calcium homeostasis in enzyme-induced hypoxic rats. METHODS： Forty-eight rats were pretreated with phenobarbital and randomly divided into six groups （eight in each group） and exposed to O2/N2/1.2 MAC anesthetics for 1 h： normal control （NC）, 21% O2/79% N2; hypoxic control （HC）, 14% O2/86% N2; normal sevoflurane （NS）, 21% O2/ N2/1.2MAC sevoflurane; hypoxic sevoflurane （HS）, 14% O2/N2/1.2MAC sevoflurane; normal halothane （NH）21%O2/79%N2/1.2MAC halothane; hypoxic halothane （HH）, 14%O2/N2/1.2MAC halothane. Liver specimens and blood were taken 24 h after exposure to calcium and determined by EDX microanalysis. RESULTS： The liver of all rats given halothane （14% O2） had extensive centrilobular necrosis and denaturation. Morphologic damage was accompanied with an increase in serum glutarnic pyruvic transminase. In groups NH and HH, more calcium was precipitated in cytoplasm and mitochondria. CONCLUSION： These results suggest that halothane increases cytosolic Ca^2＋ concentration in hepatocytes. Elevation in Ca^2＋ concentration is implicated in the mechanism of halothane-induced hepatotoxicity. sevoflurane is less effective in affecting hepatic calcium homeostasis than halothane.

Reversal of Adriamycin Resistance of Hepatocellular Carcinoma by Targeting with Recombined Adenovirus Caning Antisense mdr1 RNA

OBJECTIVE Chemotherapy is an important therapy for hepatocellular carcinoma （HCC）. However, it is not effective in many cases due to recurrence and metastasis even if the initial treatment produces a response. Multidrug resistance （MDR） is considered to be one of the considerable causes. The aim of this study was to reverse MDR of HepG2/ADM cells by blocking mdr1 with an adenovirus vector carrying antisense mdr1 in a tumor transplantated in athymic mice. METHODS PCMV IE was removed from the pshuttle vector. A 0.3 kb AFP promoter was inserted into the pshuttle vector and pCMV changed into pAFP. The pAFP and asmdr1 PCR products were doubly digested with Kpnl and Apal, the digested products were ligated by T4 ligase, the asmdr1 gene was inserted into pAFP and a newly plasmid pAFP-asmdr1 was constructed. Following digestion with PI-SceI/I-Ceu I, pAFP-asmdr1 was ligated with Adeno-X genome DNA and amplified in E.coli XL1-Blue. The HEK293 cells were transfected and virus collected. The HepG2 MDR cells （HepG2/ADM） were induced by graded resistance to ADM and were inoculated into athymic mice. After adeno-asmdr1 was injected, the expression of mdr1-mRNA and the volume of the transplantated tumor and its cells were observed. RESULTS Following injection with Adeno-asmdr1, the tumor volume in the ADM＋Adeno-asmdr1 group did not increase. However the tumor volume in the PBS plus ADM group did significantly increase （P〈0.05）. In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and was found to be reduced at 1 week and 4 weeks in the ADM＋asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM＋asmdr1 group compared to the ADM group at the 4th week （P〈0.05）. Evidence of apoptosis was observed in the tumor xenograft cells treated with Adeno-asmdr1, but there was rare or no apoptosis in the group treated with ADM and PBS. CONCLUSION Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.

IN VITRO STUDY ON CYTOTOXIC EFFECTS OF THE ORGANOPHO SPHOROUS PESTICIDE PROFENOFOS ON THE GILL CELL LINE, FG-9307, OF THE FLOUNDER (PARALICHTHYS OLIVACEUS)

The FG 9307 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.

Effect of Therapeutic Ginger on Genotoxic of Taxol Drug （Anti-Cancer） in Bone Marrow Cell of Male Mice

Medicinal use of spices/herbs has been gradually increased in the developed countries, Zingiber officinale （Ginger） is known to possess potent antioxidant and anti inflammatory properties. Therefore, the aim of this study is to determine the possible anti-mutagenic effect of ginger against the genotoxic effect of anti-cancer drug Taxol 0.6 mg/kg. This study is conducted by using two types of cytogenetic studies in bone marrow cell of mal albino mice Mus musculus （average weight 25-30 g）. The animals were randomly distributed into six groups, each of 14 mi[ce, （GI） was given the solvent, （G2） treatment of the medical dose of Taxol drug, （G3） treatment of ginger, （G4） a pre-treatment of ginger prior to treatment of drug, （G5） a simultaneous treatment of ginger and treatment of drug, （G6） a post-treatment of ginger after treatment drug. The study results show that significant increase in total chromosomal aberrations and significant increase in the number of micronuclei were observed after treatment drug. The significant structural aberrations were in the form of end-to-end associations. The numerical chromosomal aberrations were endomitosis and polyploid. The results showed that the frequencies of chromosomal aberrations and micronuclei in ginger treated group were not significantly different from control. Simultaneous treatment of ginger was found to be effective in reducing the genotoxic effects induced by drug Taxol especially in the total number of the chromosomal aberrations and the number of micronuclei.

THE ROLE OF RECOMBINANT HUMAN FUSION PROTEIN IL－6／IL－2（CH925） IN HEPADNA VIRUS INFECTION TREATMENT

The effects of CH925, a novel immune modulator, on hepadna virus infection was evaluated. Day-old ducklings were inoculated intravenously with LJ-76 DHBV containing serum. Infected ducklings were then treated with the CH925 and the mixture of IL-2 and IL-6 intravenously and the control ducklings received equivalent normal saline (NS). Blood and liver samples were taken and assayed for DHBV DNA and /or DHBsAg. At the completion of the experiment there was a inhibition of viremia with the CH925 and IL-2 + IL-6. Serum DHBV DNA was detected in 6 of 10 ducks in 100 000 unit/kg dosage group, 7 of 10 ducks in 50 000 unit/kg dosage group and 6 of 10 ducks in IL-2 + IL-6 dosage group, compared with 9 of 10 NS control, and it showed a similar result in circulating DHBsAg. When samples of liver DNA were processed for hybridization, a little difference in the DHBV DNA replication was noted between ducks receiving CH925, IL-2 + IL-6 or NS placebo. It is suggested that CH925 might be a potential remedy in HBV infection treatment.

Study on Biocompatibility of Cross-linked Hyaluronic Acid Derivatives

Objective:The cross-linked production,which was prepared by HA and cross-linking agent STMP,EDC,GP through cross-linking reaction,might be used in drug delivery system(DDS).To ensure the security of clinical application,the excellent properties such as none cell toxicity,nonirritant,none general toxicity,none immunological rejection are necessary.Methods:In accordance with the request of GB/T 16886.1 on security evaluation of medical biomaterials,cell toxicity test,hemolysis test,intracutaneous stimulation test,acute toxicity test,and hypersensitive test were required.Results:Cell toxicity of HA-STMP,HA-EDC,HA-GP were all less than 1.All hypersensitive tests were eligible.But HA-EDC,HA-GP produced different degrees of slight thrill,slight toxicity,hemolysis rate,which were larger than the standard value.Conclusion:HA-STMP possesses favourable biocompatibility,which is a kind of ideal biomaterials and drug carriers.

CUMULATIVE RESULTS OF CHEMOSENSITIVITY TEST USING MTT ASSAY IN DOUBLE-LAYER AGAROSE

Objective To investigate cumulative results of chemosensitivity test using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl （MTT） assay in double-layer agarose. Methods A total of 2 491 patients with different kinds of cancers were enrolled in the study, in which 18 kinds of different anticancer drugs were used. A computer soft was used to get charts. ResultsThe total evaluability rate was 82.7% （2 060/2 491）. Among all agents, the efficiency rates of 5-Fu, MMC, DDP, BLM and CBP were higher than the efficiency rates of others. The response rate range of different cancer in vitro sensitivity by using MTT assay in double layer agarose were from 9.2% （biliary duct） to 37.5% （malignant lymphoma）. For colon and rectum cancer, 5-Fu, DDP, MMC and BLM were more sensitive than other anti-tumor agents. For breast carcinomas, ACTD and DDP were more sensitive. For gastric cancer, 5-Fu, DDP and BLM were more sensitive. For leukemia, VM-26 and HHRT were more sensitive. ACM was more sensitive to kidney and MXT and BLM were more sensitive to pancreas cancer. For Lung cancer, DDP and EPI were more sensitive. Mean true positive rate, mean true negative rate, mean sensitivity, mean specificity and mean accuracy were 44% , 92% , 72% , 77% , and 76% , respectively. Conclusion Chemosensitivity tesing using the MTT assay in a double layer agarose was a very useful reference to chem- therapy.