糖、GA3及ABA对印度娃儿藤(Tylophora indica(Burm.f.)Merrill)体细胞胚发生的影响

从印度娃儿藤节间外植体获取愈伤组织,分析了糖、赤霉素(GA3)及脱落酸(ABA)对愈伤组织形成体细胞的影响。实验证明,含4μmol/L2,4-二氯苯氧乙酸(2,4-D)的MS培养基是获得具有成胚功能的愈伤组织的最佳培养基。在含有6μmol/L激动素(Kn)的MS培养基上,高达69%的愈伤组织分化为体细胞胚,平均单位外植体(每克愈伤组织)产胚25个。在6μmol/LKn存在的条件下,分析了蔗糖、葡糖糖对胚产生的影响,不同的糖及不同糖浓度对体细胞胚的发生影响很大。6μmol/L Kn与200mmol/L蔗糖处理胚胎发生率最大(71%),单位外植体生成49个胚。然而葡萄糖与Kn、或者葡糖糖、蔗糖与Kn三者加在一起则降低成胚率及产胚数。一定浓度的GA3和ABA能促进体细胞胚的产生。在含200mmol/L蔗糖的培养基中加10μmol/LGA3胚的生成率为98%,单位外植体产胚51个。在含200mmol/L蔗糖的培养基中加2μmol/L ABA能显著增加体细胞胚的量,该培养基上每外植体平均生成44个胚,产率为95%。本研究显示,含200mmol/L蔗糖的培养基中分别加入6μmol/L Kn、10μmol/L GA3或者2μmol/L ABA能显著提高印度娃儿藤体细胞胚发生率,而单独的葡萄糖或葡糖糖和蔗糖则有抑制作用。得到的胚均能正常发育并分化为植株。

细圆藤的化学成分研究

从细圆藤的藤部乙醇提出物中分得６个结晶，经测定理化常数和光谱数据，分别鉴定为表木栓醇，蜂蜜酸，棕榈酸，硬脂酸，白桦脂酸及胡萝卜甙。

藤黄酸对人肝癌细胞凋亡与自噬性死亡诱导作用的研究

目的观察藤黄酸对人肝癌细胞株HepG2凋亡和自噬的影响，并探讨其可能机制。方法不同浓度的藤黄酸处理H印G2细胞24h后，采用四甲基偶氮唑盐（MTT）法测定细胞增殖率，采用流式细胞术检测细胞凋亡率，采用单丹磺酰尸胺（MDC）荧光染色观察细胞内自噬泡的形成，采用weste册bJnt方法检测细胞凋亡相关蛋白Bax、bcl-2及自噬相关蛋白Beclin1的表达水平。结果藤黄酸对HepG2细胞生长的抑制作用呈剂量依赖性。O、2．0、4．0、8．0umol／L藤黄酸作用24h后，细胞凋亡率升高，分别为5．31％、29．18％、31．50％、46．09％（P〈0．05）；MDc平均荧光强度也升高，分别为6_3±1．1、82．6±4.5、132．9±15．7、157．7±9．0（P〈0．01）；促凋亡蛋白Bax表达升高，分别为0．17±0．02、O．75±0．06、0．78±0．05、O．89±0．10（P〈0．05）；抗凋亡蛋白bcl．2表达降低，分别为1．18±0．04、0．90±0．06、0．64±O．08、0．57±0．05（P〈0．05）；同时自噬相关蛋白Beclin1的表达升高，分别为0．67±0．03、0．92±0．04、0．95±0．07、1．04±0．06（P〈0．05）。结论藤黄酸通过诱导细胞凋亡与自噬性死亡的方式抑制人肝癌HepG2细胞的生长。

Triptolide protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats:Implication for immunosuppressive therapy in Parkinson’s disease

Objective Neuroinflammation with microglial activation has been implicated to have a strong association with the progressive dopaminergic neuronal loss in Parkinson＇s disease （PD）. The present study was undertaken to evaluate the activation profile of microglia in 1-methyl-4-phenyl pyridinium （MPP^＋）-induced hemiparkinsonian rats. Triptolide, a potent immunosuppressant and microglia inhibitor, was then examined for its efficacy in protecting dopaminergic neurons from injury and ameliorating behavioral disabilities induced by MPP^＋. Methods The rat model of PD was established by intranigral microinjection of MPP^＋. At baseline and on day 1, 3, 7, 14, 21 following MPP^＋ injection, the degree of microglial activation was examined by detecting the immunodensity of OX-42 （microglia marker） in the substantia nigra （SN）. The number of viable dopaminergic neurons was determined by measuring tyrosine hydroxylase （TH） positive neurons in the SN. Behavioral performances were evaluated by counting the number of rotations induced by apomorphine, calculating scores of forelimb akinesia and vibrissae-elicited forelimb placing asymmetry. Results Intranigral injection of MPP^＋ resulted in robust activa- tion of microglia, progressive depletion of dopaminergic neurons, and ongoing aggravation of behavioral disabilities in rats. Triptolide significantly inhibited microglial activation, partially prevented dopaminergic cells from death and improved behavioral performances. Conclusion These data demonstrated for the first time a neuroprotective effect of triptolide on dopaminergic neurons in MPP^＋ induced hemiparkinsonian rats. The protective effect of triptolide may, at least partially, be related to the inhibition of MPP^＋-induced microglial activation. Our results lend strong support to the use of immunosuppressive agents in the management of PD.

Apoptosis of human pancreatic cancer cells induced by Triptolide

AIM: To investigate apoptosis in human pancreatic cancer cells induced by Triptolide (TL), and the relationship between this apoptosis and expression of caspase-3' bcl-2 and bax. METHODS: Human pancreatic cancer cell line SW1990 was cultured in DMEM media for this study. MTT assay was used to determine the cell growth inhibitory rate in vitro. Flow cytometry and TUNEL assay were used to detect the apoptosis of human pancreatic cancer cells before and after TL treatment. RT-PCR was used to detect the expression of apoptosis-associated gene caspase-3' bcl-2 and bax. RESULTS: TL inhibited the growth of human pancreatic cancer cells in a dose-and time-dependent manner. TL induced human pancreatic cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of human pancreatic cancer cells with 40 ng/mL TL for 12 h and 24 h, the apoptotic rates of human pancreatic cancer cells increased significantly. RT-PCR demonstrated that caspase-3 and bax were significantly up-regulated in SW1990 cells treated with TL while bcl-2 mRNA was not. CONCLUSION: TL is able to induce the apoptosis in human pancreatic cancer cells. This apoptosis may be mediated by up-regulating the expression of apoptosis- associated caspase-3 and bax gene.

The effects of FuFangTengLiGen preparation regulating the expressions of Cx43 and E-cad of transplantation tumor of PG cell

Objective: To study the anticancer effect of Chinese compound recipe FuFangTengLiGen (FFTLG) against trans- plantation tumor of PG cell and preliminarily explore the connection with connexin43 (Cx43) and epithelial cadherin (E-cad). Methods: Model rats of transplantation tumor of human large cell cancer PG were established. The low, medium and high dose groups of FFTLG, the blank control group were set up. After 21 days medication of FFTLG through gastrogavage, the antitumor effect of each group was compared. The gene expressions of Cx43 and E-cad in each group were quantitatively detected and compared by the application of immunohistochemistry technique. Results: FFTLG could significantly inhibit the growth of transplantation tumor. FFTLG could obviously up-regulate the expressions of Cx43 and E-cad, which in the low/medium and high dose groups were separately higher than that in the blank group. Conclusion: FFTLG has definite antitumor effect against human large cell cancer and can obviously up-regulate the expressions of Cx43 and E-cad, which may be correlated with its effect of anti-tumor.

Research progress on anti-tumor properties of Marsdenia tenacissima

Tongguanteng （Marsdenia tenacissima）, which is mainly distributed in the Yunnan and Guizhou provinces of China, wasfirst recorded in Diannanbencao by Lan Mao of the Ming dynasty of China. According to recent pharmacological studies,the chemical composition of Tongguanteng （Marsdenia tenacissima） is complex and contains C21 steroidal saponins,polysaccharides, alkaloids, and other molecules, which show anti-cancer effects on various tumor cell lines. It inhibitstumor cell proliferation and growth mainly by increasing the expression of apoptosis- and cell cycle-related proteins topromote apoptosis and arrest tumor cells in the G2/M or S phase. Downregulation of the expression of vascularendothelial growth factor-2/A and matrix metalloprotease-2/9 suppresses the formation of the tumor microvasculature,leading to tumor malnutrition, increased expression of interleukin-2, glutathione peroxidase, catalase, and superoxidedismutase, and decreased interleukin-10 and malondialdehyde expression, thereby enhancing immunity andantioxidation in the body. Additionally, inhibition of epidermal growth factor receptor, hepatocyte growth factor receptor,and tyrosine-protein kinase receptor activation enhances the anti-tumor efficacy of epidermal growth factorreceptor-tyrosine kinase inhibitors as well as inhibits P-glycoprotein and cytochrome P450 to increase the concentrationof anti-tumor drugs in tumor cells.

UROTENSIN II RECEPTOR IN THE RAT AIRWAY SMOOTH MUSCLE AND ITS EFFECT ON THE RAT AIRWAY SMOOTH MUSCLE CELLS PROLIFERATION

Objective. To investigate the characteristics of urotensin II (U II) receptor in the rat airway smooth muscle and the effect and signal transduction pathway of U II on the proliferation of airway smooth muscle cells. Methods. Using 125I UII binding assay to measure the Bmax and Kd of U II receptor. Using the 3H TdR incorporation to determine the effect of U II on the proliferation of airway smooth muscle cells and its signal transduction pathway. Using Fura 2/AM to measure the effect of U II on the cytosolic free calcium concentration. Results. 1. 125I UII binding increased with the time and reached saturation at 45min. The Bmax was (11.36±0.37)fmol/mg pr and Kd was (4.46±0.61)nmol/L. 2. U II increased 3H TdR incorporation of the airway smooth muscle cells in a dose dependent manner. 3. H7, PD98059 and nicardipine, inhibitors of PKC, MAPK, calcium channel, respectively, significantly inhibited U II stimulated 3H TdR incorporation of airway smooth muscle cells. W7, inhibitor of CaM PK, had no effect. 4. Cyclosporin A, inhibitor of CaN, inhibited 3H TdR incorporation of the airway smooth muscle cells induced by U II in a dose dependent manner. 5. U II promoted cytosolic free calcium concentration increase by 18%. Conclusions. 1. There was U II receptor in the rat airway smooth muscle. 2. The effect of U II stimulated 3H TdR incorporation of airway smooth muscle cells was mediated by such signal transduction pathway as Ca2+, PKC, MAPK and CaN, etc.

Apoptosis of Hela cell induced by Celastrus orbiculatus Thunb extract and primary mechanisms

Objective： The apoptosis of Hela cells induced by ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb was studied in order to assess its antitumor effect. Methods： Hela cells were cultured in vitro and treated by a series of concentrations of ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb. Cell proliferation was detected based on MTT assay. Quantity of apoptosis were observed and analyzed by flow cytometry with Annexin V and propidium iodide double staining. P53 gene expression was detected by flow cytometry. Results： The proliferation of Hela cells was obviously inhibited by 15, 30, 60 and 120 IJg/mL extract of Celastrus orbiculatus Thunb and apoptosis of Hela was induced by dosed dependent manner. P53 gene showed increasing tendency when treated by 60-480 IJg/mL extract of Celas- trus orbiculatus Thunb. Conclusion： The ethyl acetate extract and n-butanol extract of Celastrus orbiculatus Thunb could induce apoptosis of Hela gastric cancer cells by dose dependent manner, which maybe one of the important mechanisms of Celastrus orbiculatus Thunb＇s anticancer effects. P53 protein expression in Hela was up-regulated by Celastrus orbiculatus Thunb, which maybe one of the molecular mechanisms involved in the anticancer and proapoptotic effect of Celastrus or- biculatus Thunb.

The cellular labeling and pH-sensitive responsive-drug release of celastrol in cancer cells based on Cys-CdTe QDs

As one of the active compounds derived from Traditional Chinese Medicine,Celastrol(CSL)had cytotoxicity for human leukemia cancer cells K562 and its multidrug-resistant cell line K562/A02.Here,we introduced cysteamine-modified CdTe QDs as the labeling and drug carrier into CSL research and found that the self-assembly and conjugation of anticancer molecular CSL with the Cys-CdTe QDs could significantly increase the drug’s cytotoxicity for K562 cells.More important,these CSL-Cys-CdTe nanocomposites could overcome the multidrug resistance of K562/A02 cells and efficiently inhibit the cancer cell proliferation by realizing the pH-sensitive responsive release of CSL to cancer cells.The enhanced cytotoxicity was caused by the increase of the G2/M phase arrest for K562/A02 cells as well as for K562 cells.Cys-CdTe QDs can readily bind on the cell plasma membranes and be internalized into cancer cells to trace and detect human leukemia cancer cells in real time.In addition,these Cys-CdTe QDs can facilitate the inhibition of the multidrug resistance of K562/A02 cells and readily induce apoptosis.As a good photosensitizer for the therapy,labeling,and tracing of cancer cells,the combination of CSL with Cys-CdTe QDs can optimize the use of and a new potential therapy method for CSL and yield new tools to explore the mechanisms of active compounds from Traditional Chinese Medicine.

Celastrus orbiculatus extract induces mitochondrial-mediated apoptosis in human hepatocellular carcinoma cells

OBJECTIVE： To investigate the apoptotic effects and underlying molecular mechanisms of Celastrus orbiculatus （C. orbiculatus） extract in human hepa- tocellular carcinoma cells. METHODS： Human hepatocellular carcinoma cells （HCCLM6） were treated with C. orbiculatus extract （COE） at different nontoxic concentrations （10, 20, 40, 80, and 160 IJg/mL）. The effect of COE on HC-CLM6 viability was examined using 3-（4,5-dimethyl- thiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assays. Cellular apoptosis following COE treatment was assessed by flow cytometry and western blot analysis. RESULTS： COE significantly inhibited cell viability and induced apoptosis of HCCLM6 cells in a dose-dependent manner. Apoptosis was accompa- nied by increased Bax expression and decreased Bcl-2 expression. In addition, COE treatment led to the release of cytochrome c, activation of cas- pase-3, and cleavage of poly （ADP-ribose） poly- merase （PARP）. Furthermore, activation of extracel- lular signal-regulated kinase （ERK）, p38 kinase, and c-Jun N-terminal kinase （JNK） phosphorylation, and down-regulation of Akt phosphorylation was ob- served. CONCLUSION： COE induces mitochondrial-mediat- ed, caspase-dependent apoptosis in HCCLM6 cells, which might be attributed to the activation of mito- gen-activated protein kinase （MAPK） and inhibition of Akt signaling pathways. These data suggest that COE may be a potential treatment for human hepa- tocellular carcinoma.

Cytotoxicity of anti-tumor herbal Marsdeniae tenacissimae extract on erythrocytes

Marsdeniae tenacissimae extract （MTE） has been used as an adjuvant medicine for cancer therapy for a long time. Although massive studies demonstrated its considerable anti-cancer effect, there is no research on its influence on erythrocytes, which are firstly interacted with MTE in the circulation. To investigate the influence of MTE on erythrocytes, we used a flow cytometer to detect the MTE-treated alternations of morphology, calcium concentration, and reactive oxygen species （ROS） level in erythrocytes. We used hemolysis under different osmotic solutions to evaluate the fragility of erythrocytes. Data showed that MTE treatment dose-dependently increased the ratio of erythrocyte fragmentation （P〈0.001） and shrinking, and elevated the forward scatter （FSC） value （P〈0.001） and calcium accumulation （P〈0.001）. MTE induced ROS production of erythrocytes under the high glucose condition （P〈0.01） and consequently caused a rise in fragility （P〈0.05）. These results suggest that MTE induces cytotoxicity and aging in erythrocytes in a dose-dependent manner, and presents the possibility of impairment on cancer patients＇ circulating erythrocytes when MTE is used as an anti-cancer adjuvant medicine.