【作者】 何海涛；

【导师】 洪敏；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 海洛因是滥用最为广泛的毒品,成瘾最快,毒性最烈。而对神经系统作用尤为明显。本实验室之前已经证实,达到一定浓度的海洛因可以抑制C6细胞增殖,促进ADA基因的转录。脑组织中胶质细胞是神经元细胞的10倍以上,对神经元细胞起着营养支持等作用。本实验采用C6神经胶质瘤细胞为靶细胞,通过MTT实验,利用特异性阿片受体阻断剂,研究海洛因抑制细胞生长的受体机制。给予海洛因同时补偿腺苷酸鸟苷酸,研究是否因此减弱了海洛因对细胞增值的抑制作用。进行C6细胞的体外培养,给予海洛因同时补偿腺苷酸鸟苷酸,提取作用后细胞的总RNA,并采用RT-PCR法检测ADA酶的mRNA水平转录物含量的相对变化,希望借此了解海洛因对嘌呤核苷酸分解代谢的影响,为阿片类药物作用机理的研究增加新的内容,并期望在阿片类镇痛药物的戒断治疗方面提供一些理论根据。本研究结果显示:1.MTT实验证实mu受体与detal受体阻断剂不能完全的阻断海洛因对C6细胞的增殖抑制作用,但是加大阻断剂剂量可以有效的减少海洛因的细胞增殖抑制。细胞数量的改变主要来自海洛因对细胞增殖的抑制。补偿腺苷酸鸟苷酸后的C6细胞,没有明显对抗海洛因造成的细胞增殖抑制。2.ADA转录物含量在海洛因合并补充腺苷酸鸟苷酸后有不同程度的降低,并且补充的腺苷酸鸟苷酸量越大,ADA转录物含量降低的越大。以上结果表明,海洛因对细胞增殖的抑制作用,并非由mu和detal受体单一控制,但是受体参与了海洛因对细胞的抑制作用。同时补充腺苷酸和鸟苷酸可降低C6细胞分解代谢关键酶ADA的基因表达。更多还原

【Abstract】 The abuse and addiction of opiates is a serious social and medical problem, which threatens the health and happiness of people. The chronic exposure to opiates such as morphine and heroin can cause psychological and physical dependence to addicts, it’s an important subject to study the basis of addiction and dependence.At present, there are little research studying the relationship of the dependence of opiates and receptor antagonist as well as add adenosine. We inject sensitive antagonist into receptor, cultivate it in vitro cell with heroin, to find whether heroin has inhibitory effects on the cell proliferation using the MTT colorimetry method. In addition, we test the antagonist NTI on the c6cell which effected by heroine for 24h, and examine the influence of antagonist on the cell. Opioid drugs caused the excessive decomposition of adenosine, and there’s an obvious change on the enzyme’s activity and gene expression which is related to adenosine metabolism. Some literatures show the opiate addiction may be related with adenosine metabolism, so we add adenosine and guanosine to heroin, and examine the influence. We choose the ADA pathway as study object which has a strong metabolic process, observe the effect on the inhibition of cell proliferation as well as the gene expression of ADAmRNA.The experiment on receptor antagonist is divided into four parts: the cyprodime of mu-receptor-antagonist 1h then heroin 24h; the NTB of detal-receptor-antagonist 1h then heroin 24h; the NTB of detal-receptor-antagonist 1h then morphine 24h; the heroin 24h then the NTI of detal-receptor-antagonist 24h. there are control group and heroin(morphine) group in every part, the antagonist with four dose: 1nmol/L ,10n mol/L, 100nmol/L, 1000nmol/L were given for 1h then heroin was added. While the NTI was given 20ug/ml for 24 then antagonist was added. We choose the heroin with the concentration of 20ug/ml, concluded from the experiment at which it inhibits the proliferation of c6 cell significantly without leading to the cell death.The experiment of MTT confirms the heroin inhibition of cell proliferation is not merely through a receptor, the antagonist weakens the effect of heroin on c6 glioma cells in inhibiting cell proliferation, and there’s an increasing trend with the dose increase, there’re marked difference among the cyprodime of mu-receptor-antagonist group, the NTB of detal-receptor-antagonist group, the NTI of detal receptor antagonist group and the grads group of NTI with the concentration of 1000nmol/L, 1nmol/L. there’s marked difference only in the NTB group with the concentration of 100nmol/L and 10nmol/L in the neighbor concentration group of antagonist. The inhibition effect of heroin and antagonist on cell proliferation and DNA synthesis may be caused by the adverse reaction, which means that the drug leads to the cell death. To exclude the possibility, we determine the cell vigor by trypan blue method. The results indicate that there is no significant deviation between control and heroin group and cell vigor are all over 90 percent, which confirms that the decrease of cell growth is not caused by cell death.Therefore, we believe that heroin results in the inhibition effect not through a single opiates receptor. At present, we study only with the mu-receptor and detal-receptor, we will make further research on other receptors, and observe the gene expression related to cell proliferation, finding heroin’s inhibition effect on C6 glioma cell proliferation sensitively.The achievement of adenosine catabolism is through the pathway of adenosine deaminase ADA and adenosine kinase AK. ADA’s effect on adenosine catabolism is superior to AK. ADA is the key enzyme of purine nucleotide catabolism, it catalyzes the adenine nucleotide into hypoxanthine nucleoside, and then decompose into hypoxanthine. In this experiment, we compare the group of added AMP, GMP with the group of heroin in c6 cell, the expression level of ADAmRNA lowered, and an increasing dose of AMP,GMP will weaken the effect of heroin on the expression level of ADAmRNA. We observe the inhibition effect on cell proliferation with the adenosine and guanosine added using the MTT colorimetry method. but not found pattern changes, which may be the result of that gene expression behaves more obviously than cell proliferation in short time.There is no obvious difference on the expression level of ADAmRNA between the group with high dose adenosine and guanosine added and the control group. The ADA/β-actin1 in control group is 0.4877, while 0.5009 in the group with adenosine, 10.7270 in the group with heroin, 0.5227 in the group with heroin and high dose adenosine and guanosine(100ug/ml), 0.5830 in the group with heroin and low dose adenosine and guanosine(25ug/ml).In conclusion, the mu-receptor and detal-receptor that we choose effect by antagonist and then heroin(morphine), the cell proliferation is weakened, and there’s a similar circumstance as the heroin is firstly given. Though it weakens the damage to the cell by the opiates, there is still a marked difference compared with the control group, the inhibition of cell proliferation decreases more in the high dose antagonist compared with the low dose group. With the decrease in the concentration of antagonist, the inhibition of cell proliferation is more obvious, there is usually no marked difference between the neighbor group. The c6 cell treated with heroin and then AMP,GMP can weaken the catabolism of adenosine, behaves in that the mRNA expression of the metabolism key enzyme ADA decreases compared with the one simply giving heroin. The more the AMP and GMP is added, the smaller the mRNA expression of ADA is. There is no obvious change between the group treated only with AMP and GMP and the control group.更多还原