目的 探讨重楼(Paris polyphylla)的醇提取物活性单体pp-22对人鼻咽癌CNE-1细胞的增殖、 周期和凋亡的影响.方法 采用MTT法,流式细胞术实验检测pp-22对人鼻咽癌CNE-1细胞的增殖、 周期及凋亡的影响.结果 重楼提取物活性单体pp-22能抑制CNE-1细胞的生长,并与时间、 剂量呈正相关;流式细胞术检测显示,pp-22能导致人鼻咽癌CNE-1细胞发生G2/M期周期阻滞和凋亡.结论 重楼醇提取物活性单体pp-22能够抑制CNE-1细胞增殖,引起G2/M期周期阻滞,诱导凋亡.

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.

Objective To determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes. Methods The cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined. Results NE at a concentration of 50 μ mol/L induced an obvious increase in the activity of LDH in the culture medium ( P <0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 μ mol/L Ber ( P <0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner ( P <0.01). Moreover, Ber at a dose of 2 μ mol/L markedly decreased the ROS and TNF-α productions ( P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE ( P <0.05)h. Conclusion The present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Aim: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. Methods: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. Results: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. Conclusion: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

BACKGROUND:Sodium valproate inhibits proliferation in neuroblastoma and glioma cells, and inhibits proliferation and induces apoptosis in hepatoblastoma cells. Information describing the molecular pathways of the antitumor effects of sodium valproate is limited; therefore, we explored the mechanisms of action of sodium valproate in the human hepatoblastoma cell line, HepG2.METHODS:The effects of sodium valproate on the proliferation of HepG2 cells were evaluated by the Walsh-schema transform and colony formation assays. Sodium valproate-induced apoptosis in HepG2 cells was investigated with fluorescence microscopy to detect morphological changes; by flow cytometry to calculate DNA ploidy and apoptotic cell percentages; with Western blotting analyses to determine c-Jun N-terminal kinases (JNK), p-JNK, Bcl-2, Bax, and caspase-3 and -9 protein expression levels; and using JC-1 fluorescence microscopy to detect the membrane potential of mitochondria. Statistical analyses were performed using one-way analysis of variance by SPSS 13.0 software.RESULTS:Our results indicated that sodium valproate treatment inhibited the proliferation of HepG2 cells in a dose-dependent manner. Sodium valproate induced apoptosis in HepG2 cells as it: caused morphologic changes associated with apoptosis, including condensed and fragmented chromatin; increased the percentage of hypodiploid cells in a dose-dependent manner; increased the percentage of annexin V-positive/propidium iodide-negative cells from 9.52% to 74.87%; decreased JNK and increased phosphate-JNK protein expression levels; reduced the membrane potential of mitochondria; decreased the ratio of Bcl-2/Bax; and activated caspases-3 and -9.CONCLUSION:Sodium valproate inhibited the proliferation of HepG2 cells, triggered mitochondria-dependent HepG2 cell apoptosis and activated JNK.

Traditional teaching mode of biochemistry experiment couldn’t satisfy the demand of cultivating creative medical talents in new era. Offering designing experiment is the developing direction of experiment teaching reforms. But in the practice,there are various of obstacles which prevent the reforming process.

Objective To observe the influence of hepatocyte growth-promoting factor (pHGF) on monocyte chemotatic protein-1-(MCP-1) and aristolochic acid Ⅰ(AAⅠ)-induced epithelial-to-mesenchymal transition (EMT) and apoptosis of human kidney epithelial cell line(HKC).Methods The HKC cells were randomly divided into blank control group (control groups), epithelial-to-mesenchymal transition model group (model group),and pHGF inhibition group (pHGF groups) with pHGF at different concentrations (0.15,1.5,15,150,and 1 500 ng/ml).The EMT model was established by exposing HKC cells to MCP-1(0.1 μg/ml)and AAⅠ(10 μg/ml).Cells in the pHGF groups were the model cells treated with different concentrations of pHGF.Cells in the control group were cultured routinely.WST-8 method and flow cytometry were used to observe the proliferation and apoptosis of HKC cells,respectively.The mRNA expression of α-smooth muscle actin(α-SMA) was determined by reverse transcriptase-polymerase chain reaction(RT-PCR),and the expression of α-SMA,fibronectin (FN),and transforming growth factor-β1(TGF-β1) in HKC cells were assessed by indirect enzyme immunohistochemistry.Results The cell inhibitory rate,apoptotic rate,and expression of α-SMA mRNA were significantly increased in the model group and pHGF groups compared with those in the control group (P<0.01),indicating the successful establishment of EMT model.Compared with the model group,pHGF at 150 ng/ml,but not at other concentrations,significantly decreased the inhibition rate of HKC cells(P<0.01).The apoptotic rate of HKC cells in all the pHGF groups were significantly lower than that in the model group (P<0.01).pHGF at 150 ng/ml also greatly decreased the expression of α-SMA mRNA,and significantly down-regulated the expression of α-SMA,TGF-β_1,and FN protein.Conclusion pHGF at 150 ng/ml can partly reverse MCP-1- and AA Ⅰ-induced HKC cell growth inhibition,apoptosis,and EMT.

目的研究结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人肾小管上皮细胞（HKC）转分化的影响。方法用巨噬细胞趋化因子-1（MCP-1）和马兜铃酸Ⅰ（AAⅠ）联合诱导HKC转分化模型。实验设空白对照组、模型对照组、PEI组、CTGFASODN10ng/mL组和CTGF ASODN100ng/mL组,分别采用WST-8法、流式细胞术、RT-PCR法、细胞免疫组织化学法检测不同浓度的CTGF ASODN对HKC转分化模型细胞增殖和细胞周期的影响,以及α-SMA mRNA表达和α-SMA、TGF-β1、Fibronectin（FN）蛋白表达的变化。结果 100ng/mL的CTGF ASODN对MCP-1和AAⅠ联合诱导HKC转分化细胞具有促进细胞增殖的作用,并可减少实验模型导致的细胞S期阻滞,降低α-SMA mRNA表达,减少α-SMA、TGF-β1和FN蛋白的表达。结论 CTGFASODN能够抑制MCP-1和AAⅠ导致的人HKC转分化。

For example,a case of fast DNA extraction and PCR analysis of rabbit's CollagenαⅧ gene as a Method for Comprehensive Biochemical Experiments,elaborated on the comprehensive test of the experimental program,to analyze the experimental results obtained on the biochemical synthesis experiment teaching were discussed.

目的:研究聚乙烯亚胺(PEI)-PKC-α反义脱氧核寡酸(ASODN)对人肝癌SMMC-7721细胞的增殖抑制作用.方法:RT-PCR检测肝癌SMMC-7721细胞、HepG2细胞PKC-α mRNA表达的差异,细胞增殖抑制实验检测PEI和ASODN混合的最佳质量比,WST法和克隆形成抑制实验检测细胞增殖抑制作用,免疫荧光检测PKC-α的表达水平.结果:SSMC-7721细胞PKC-α mRNA表达相对较高,PEI和ASODN的质量比为0.75/1时是PEI转染反义核酸的合适比例,对SMMC-7721细胞具有明显的增殖克隆抑制作用,且呈剂量-效应关系;ASODN和PEI-ASODN对SMMC-7721的IC50分别为16.6 μmol/L和0.58 μmol/L;PEI-ASODN能够有效抑制PKC-α蛋白的生物合成.结论:PEI-ASODN能显著抑制SMMC-7721细胞增殖和克隆形成,下调PKC-α蛋白的表达.

BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity, which has an extensive application in gene therapy for many diseases. However, the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN, TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology, and Department of Pediatric, the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy, natal infant. Informed consent was obtained from his parents, and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate, mean fluorescence intensity of FAM marked CpG ODN, expressions of MNCs, proliferations of lymphocytes and the IgG levels of culture supematants were detected by flow cytometry, fluorescence techniques, MTT assay and ELISA, respectively. MAIN OUTCOME MEASURES: The uptake rate, the mean fluorescence intensity of CpG ODN by MNCs, subgroups and proliferations of lymphocytes, and IgG levels of culture supematants. RESULTS: Compared to the pure CpG ODN group, the uptake rate of the conjugated complexes group was higher (98%), the peak level of up-taking occurred earlier, and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+, CD22+, and CD20+ was increased, A value and IgG levels in supematants were all higher than that of the control group. CONCLUSION: CD40 tigand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte, enhancing its immunological efficiency.

Biochemistry experiment is an important means to train students to master basic knowledge and skills of biochemistry.This article discusses how to improve the teaching quality of biochemical experiment teaching.

随着生命科学的迅猛发展,生物化学与分子生物学的理论和实验技术已经被广泛应用到基础医学、临床医学及药学等各领域的研究中,是培养新世纪医学人才的必备技能和手段。生化实验教学在培养有知识、有技能等高素质创新型人才方面占有更重要的地位。为了实现创新型人才的培养目标,本教研室对现有的实验教学模式进行改革。本文在实践基础上,对实验教学内容及实验室管理与建设各方面的改革进行阐述与探讨。

Objective To investigate the effects of antisense oligonucleotides(ASODN)targeted protein kinase Cα(PKCα)on apoptosis of A549 cells.Methods PKCα ASODN and random oligonucleotides(RODN)were transfected into A549 cells mediated by polyethyleneimine(PEI).①A549 cells were divided into control group,PEI group,PEI-ASODN(1.25,1.50 μmol/L)groups,and the morphology was examined by fluorescence microscope and transmission electron microscope.②A549 cells were divided into control group,PEI group,PEI-RODN(1.50 μmol/L)group,PEI-ASODN(1.00,1.25,1.50 μmol/L)groups.Then the percentage of hypodiploid cells of A549 cells treated with PKCα ASODN for 48 h was determined with propidium iodide(PI)staining by flow cytometry,and the early apoptosis rate was detected with Annexin Ⅴ-FITC and PI dual parameter by flow cytometry.Results Nuclear fragmentation and condensation were observed in A549 cells treated with PKCα ASODN.The percentages of hypodiploid cells and early apoptosis rates increased dose-dependently in PEI-ASODN groups,significantly higher than in control group,PEI group and PEI-RODN group(P0.05).Conclusion The PKCα ASODN mediated by PEI could induce the apoptosis of A549 cells.

Aim:To investigate the proliferative inhibition effect of antisense oligonucleotides(ASODN) targeting for protein kinase C alpha(PKC-α) mediated by polyethyleneimine(PEI) on hepatocelluar carcinoma SMMC-7721 cells. Methods: The difference of PKC-α mRNA expression on hepatocelluar carcinoma SMMC-7721 cells and HepG2 cells was detected by reverse transcription-polymerase chain reaction(RT-PCR).The best qualitative ratio of PEI/ASODN was determined by proliferative inhibition on SMMC-7721 cells.PKC-α ASODN were transfected with PEI,the proliferation and the clone formation of SMMC-7721 cells were detected by WST method and clone formation assay,respectively,and the expression of PKC-α was analyzed by immunofluorescence test.Results: The expression of PKC-α mRNA was higher in SMMC-7721 cells than that in HepG2 cells,the best qualitative ratio of PEI /ASODN on SMMC-7721 cells was at 0.75/1.Compare with the control group,PEI group,the proliferation and clone formation rates of SMMC-7721 cells treated with PEI-ASODN were significantly inhibited(P<0.05),and the inhibitive effect was much more significant when the dose of PEI-ASODN increased.The IC50 of ASODN on SMMC-7721 cells' proliferation was 16.6 μmol/L and the IC50 of PEI-ASODN on SMMC-7721 cells was 0.58 μmol/L.And the expression of PKC-α protein in SMMC-7721 cells treated with PEI-ASODN was significantly lower than those in the control group detected by immunofluorescence test.Conclusion:The PKC-α ASODN mediated by PEI can inhibit the proliferation and clone formation in SMMC-7721 cells significantly and can down-regulate the expression of PKC-α protein.

BACKGROUND: CpG oligodeoxynucleotide (ODN) is a type of highly effective immune adjuvant with low toxicity,which has an extensive application in gene therapy for many diseases. However,the specificity for species and cells leading to low uptake by cells and degradation by nuclease blocks its clinical application. OBJECTIVE: To explore the specific delivery and its immunologic efficacy of CpG ODN targeting B lymphocytes of umbilical cord blood by CD40 ligand-receptor-mediated carrier system. DESIGN,TIME AND SETTING: An observation and control experiment was performed at the Department of Hematology,and Department of Pediatric,the First Affiliated Hospital of Jinan University from April 2004 to October 2007. MATERIALS: Fresh umbilical cord blood with heparin was obtained from healthy,natal infant. Informed consent was obtained from his parents,and the experiment was approved by the hospital Ethics Committee. METHODS: CD40 ligand (CD40L)-EDC-PLL-CpG ODN conjugated complex was prepared. Mononuclear cells (MNCs) from umbilical cord blood were co-cultured with conjugated complexes. Uptake rate,mean fluorescence intensity of FAM marked CpG ODN,expressions of MNCs,proliferations of lymphocytes and the IgG levels of culture supernatants were detected by flow cytometry,fluorescence techniques,MTT assay and ELISA,respectively. MAIN OUTCOME MEASURES: The uptake rate,the mean fluorescence intensity of CpG ODN by MNCs,subgroups and proliferations of lymphocytes,and IgG levels of culture supernatants. RESULTS: Compared to the pure CpG ODN group,the uptake rate of the conjugated complexes group was higher (>98%),the peak level of up-taking occurred earlier,and intracellular fluorescence intensity maintained much more stable. Expressions of CD19+,CD22+,and CD20+ was increased,A value and IgG levels in supernatants were all higher than that of the control group. CONCLUSION: CD40 ligand-receptor-mediated carrier system is helpful for CpG ODN delivery targeting to B lymphocyte,enhancing its immunological efficiency.

Objective To investigate the effects of siRNAs targeted protein kinase C α(PKCα) on the proliferation and apoptosis of A549 cell line.Methods Six PKCα siRNAs were designed and chemical synthesized. Candidate PKCα siRNA was transfected into A549 cells,PKCα mRNA level was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Then,the effects of more effective PKCα siRNAs on A549 were further investigated by using clone formation assay,Hoechst 33258 staining,propidium iodide (PI) staining,Annexin V-FITC and PI dual staining and western blot. Results Six candidate PKCα siRNAs were designed and named as No.1,No.2,No.3,No.4,No.5 and No.6 PKCα siRNA. Compared with controls,PKCα mRNA levels were all significantly downregulated and the cell proliferation was inhibited in A549 cells treated with candidate PKCα siRNAs except No.5 (P0.05).The No.3,No.6 and No.1 PKCα siRNAs,which inhibited A549 cell growth and PKCα expression more effectively,were identified among 6 candidate PKCα siRNAs. Compared with controls,the clone formation was significantly inhibited (P0.05),percentages of hypodiploid cells and early apoptosis rates were significantly increased (P0.01),and the expression of PKCα protein was significantlydecreased in A549 cells treated with No.3,No.6 or No.1 PKCα siRNA(P0.01). In addition,the typical morphologic changes of apoptosis were found in PKCα siRNA-treated A549 cells. Conclusion Three PKCα siRNAs in six candidate PKCα siRNAs can effectively downregulate PKCα protein expression,induce apoptosis and inhibit proliferation of A549 cells.

OBJECTIVE:To investigate the effects of antisense oligonucleotides (ASODN) targeting protein kinase C alpha (PKCalpha) on the proliferation of A549 cells.METHODS:PKCalpha ASODN and random oligonucleotides (RODN) were transfected into A549 cells mediated by polyethyleneimine, and the proliferation and clone formation of A549 cells were detected by CCK-8 and clone formation assay, respectively. The expression of PKCalpha in the transfected cells was analyzed by RT-PCR and Western blotting.RESULTS:Compared with those in the control group, PEI group and PEI-RODN group, the proliferation and clone formation of A549 cells treated with ASODN targeting PKCalpha were significantly inhibited (P<0.05). The expressions of PKCalpha mRNA and protein in PKCalpha ASODN-transfected A549 cells were significantly lower than those in the other 3 groups (P<0.05).CONCLUSION:The PKCalpha ASODN mediated by PEI down-regutates the expression of PKCalpha gene and suppress the proliferation and clone formation of A549 cells.

AIM: To design,prepare and screen out functional small interfering RNA(siRNA) for specifically silencing proliferating cell nuclear antigen(PCNA) gene expression and effectively inhibiting cell proliferation on human hepatoma cell line SMMC-7721,human gastric carcinoma cell line SGC-7901 and human colorectal carcinoma cell line Caco2.METHODS: PCNA siRNA was designed based on previous studies about design guidelines and synthesized in vitro by T7 Mega short script reaction kit according to the manufacturer's instructions.After purification,the integrities of siRNA were identified through 19% denaturing polyacrylamide gel electrophoresis,and the concentrations of the generated siRNA were measured by testing the absorbance at 260 nm using a spectrophotometer.Four synthesized double-strand siRNA were transfected into three types of carcinoma cell lines by LipofectamineTM2000 reagent,respectively.WST-8 assay was employed to examine the proliferative inhibitions of these three cell lines.The subsequent alterations on PCNA mRNA and protein levels were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochemistry,respectively.RESULTS: 3 sequences,No.2,No.3 and No.4 PCNA siRNA showed effective inhibitions on tumor cells among the 4 candidate siRNA,and a single dose of 50 nmol/L of No.2,No.4 PCNA siRNA showed the most effective inhibitory rates as more than 62% at 48 h after transfection.50 nmol/L of No.2 and No.4 PCNA siRNA transfection caused 72% decrease of PCNA mRNA level and almost completely loss of the protein in human Caco2 cells.CONCLUSION: No.2 and No.4 PCNA siRNA have been screened out in this study,which show the capability to effectively down-regulated PCNA expression and significantly inhibit the proliferation of carcinoma cells.The optimal concentration is 50 nmol/L and satisfactory effects are achieved 48 h after transfection in vitro.