细胞工程研究进展
细胞工程研究进展
名词解释
1. 细胞工程:是应用细胞生物学和分子生物学的方法,按照一定的实验设计方案,在细胞整体、亚细胞结构或者组织水平上, 改变细胞内的遗传物质,获得重构的细胞、组织、器官以及个体的新型生物或特种细胞产品的综合性生物工程技术. 即通过细胞培养、细胞融合、细胞核移植、染色体操作与转基因等实验技术来实现.
组织工程是应用细胞生物学和工程学原理, 将人体某部分的组织细胞种植和吸附在一种生物材料的支架上进行人工培养繁殖, 扩增, 然后移植到人体内所需要的部位, 从而达到器官修复或再造的治疗目的的一种技术.
2. 干细胞(Stem cells):是一类具有自我更新和分化潜能的细胞。它包括胚胎干细胞和组织干细胞。胚胎干细胞可以发展成为完整的个体,而组织干细胞又可细分为多能干细胞和单能干细胞,定向分化为细胞或组织
3. 组织工程的概念:组织工程是应用细胞生物学和工程学原理, 将人体某部分的组织细胞种植和吸附在一种生物材料的支架上进行人工培养繁殖, 扩增, 然后移植到人体内所需要的部位, 从而达到器官修复或再造的治疗目的的一种技术.
4. 死亡受体(TNFR 、FasR 、DR3、DR4、DR5)是一类通过与相应配体结合,传递细胞凋亡信号的跨胞膜蛋白。
5.Gene therapy (基因治疗): used to replace genes that have been lost or whose expression is low resulting in the accumulation of malignancies (恶性)
6.FAS: FAS 又称APO-1或CD95, 属于TNF 受体超家族。FAS 分布于胸腺细胞、激活淋巴细胞和部分肿瘤细胞等的细胞膜, 少量以可溶性形式存在于胞质和血清中。
FASL:FASL 又称FAS 配体,是一种分子量约为40KD 的Ⅱ型跨膜蛋白,属TNF 家族成员。FAS 是FASL 的受体,具有细胞表面跨膜结构
8. FADD: FADD 又名MORT1, FAS 相关死亡域蛋白。FADD 蛋白是一种相对分子质量为23 kD胞浆蛋白, 其基因定位于人染7. 色体11q13.3上, 由两个外显子和一个内含子组成, 编码208个氨基酸。FADD 蛋白由羧基末端的死亡结构域( death domain,DD) 和氨基末端的死亡效应域( death effect domain, DED)两部分组成。
FLIP:Fas 相关死亡区域蛋白样白介素-1β转换酶抑制蛋白(FLICE-inhibitory protein, FLIP) 是近年来发现的一类含有死亡效应域( death effect domains, DED) 的凋亡抑制蛋白 9.
10. caspase: caspase( cysteine-containing aspartate-spicific protease)意为含天冬氨酸特异性半胱氨酸蛋白酶, 是细胞凋亡的主要执行者。在caspase 家族中, caspase-8是重要的启动型caspase, 可活化caspase-3等效应型caspase, 使细胞凋亡。哺乳动物caspase 的活化主要有细胞外(caspase-8途径/非线粒体途径, 也被称为死亡受体通路) 和细胞内( caspase-9途径/线粒体途径) 两条途径, 且这两条途径有交叉。
11. 表达载体:实验室中使用的用于过度表达糖基转移酶的载体,, 是一种对于卢卡斯等人所用的载体修改而来, 这些质粒包括在5’-接合工体和3’-接合受体位点间包含了博罗霉素的选择性标记位点
12 IgG:是主要的免疫球蛋白,约占人体血浆中丙种球蛋白的70%,分子量约为15万,其分子由四条肽链组成,共有四个亚型,分别为1,2,3,4,在血清中的浓度由高到低的次序为1〉2〉3〉4,
Ps :需要注意的是人体内共有五大抗体细胞:IGA,IGM,IGG ,IGD 和IGE
13.糖蛋白:由比较短,往往带分支的寡糖与多肽链某些特殊部位的羟基或酰胺基共价连接而形成的一种结合蛋白质
14. 糖基化工程:在深入研究糖蛋白中糖链结构与功能关系的基础上,通过人为的改变(包括增加和删除),调整糖蛋白的表面的糖链而达到的改变糖蛋白的生物学功能的目的
15. 级联反应: 通过多次的逐级放大使较弱的输入信号转变为极强的输出信号,导致各种生理响应的过程
16. RNA 干扰:(RNA interference ,RNAi) 是正常生物体内抑制特定基因表达的一种现象,它是指当细胞中导入与内源性mRNA 编码区同源的双链RNA(double stranded RNA,dsRNA) 时,该mRNA 发生降解而导致基因表达沉默的现象,这种现象发生在转录后水平,又称为转录后基因沉默(post-transcriptional gene silencing,PTGS) 。外源dsRNA 进入细胞后产生的小分子干扰RNA (small interfering RNA, siRNA)的反义链和多种核酸酶形成了沉默复合物(RNA-induced silencing complex,RISC) ,RISC 具有结合和切割mRNA 的作用而介导RNA 干扰的过程。RNAi 具有特异性和高效性。这种技术已经成为研究基因功能的重要工具,并将在病毒病、遗传性疾病和肿瘤病的治疗方面发挥重要作用。依据RNA 干扰现象, 科学家建立了RNA 干扰技术, 即人为设计合成针对某特定基因序列的dsRNA 来关闭或抑制该基因的表达。RNA 干扰已被证实是一种特异、高效、经济的使基因表达受抑的技术手段。
缩写:MCL-1 human myeloid differentiation protein 人类骨髓分化蛋白
TNF tumor necrosis factor 肿瘤坏死因子
IAP (inhibitor of apoptosis protein ) 细胞凋亡蛋白抑制因子
Bik (bcl-2 interacting killer protein)
Bcl-2 proto-oncogene (第一致癌基因)
NGF nerve growth factor (神经生长因子)
EGF epidermal growth factor
SCR stem cell factor
IGFs insulin like growth factors
EPO erythropoietin
CARD caspase recruitment domain
DED death effector domain
SnRNA 小核RNA
ShRNA 小发夹rna 干扰技术
SiRNA 小干扰RNA 干扰技术
HPAEC 高效阴离子交换色谱
XIAP x 染色体连锁的凋亡抑制蛋白
Chapter1
1.Activation of Caspases(细胞凋亡蛋白酶的激活)
◆ TNF-type of receptors that engage cell death pathways include Fas/Apo1/CD95 type 1
tumor necrosis factor α (TNFα) receptor (TNFR1) (specific for TNF α), death receptor 3 (DR3; specific for the Apo3 ligand), and DR4 as well as DR5 (both of which bind Apo2/TRAIL). All of these receptors induce cell death by acting as scaffolds for caspase activation(所有的这些凋亡因子在诱导细胞凋亡中扮演了细胞蛋白酶激活的桥梁作用)
◆ The best-characterized pathway of caspase activation is the processing of procaspase-8 to caspase-8 triggered following binding
of FasL to the death receptor Fas/Apo1/CD95.
在细胞凋亡蛋白酶激活的过程中最有特征的通路是由前体蛋白酶8到细胞凋亡蛋白酶8的转化过程,这个过程需要将前体蛋白酶上连上Fasl ,以及具有死亡信号的受体蛋白Fas/Apo1/CD95,
Fas/Apo1/CD95 exists as a preassociated complex which appears to be required for functional apoptosis signaling following FasLbinding. When FasL binds to its cognate receptor Fas/Apo1/CD95, the death domain (DD), an intracellular portion(细胞内的部分) of the Fas/Apo1/CD95 receptor of about 90 amino acids, interacts with the death domain of a bipartite adaptor molecule called FADD in a homotypic interaction. The rapid recruitment of FADD (DD of an adapter molecule ) to form these receptor complexes is associated with the formation of microaggregates. The N-terminal death effector domain (DED) of FADD in turn interacts with the DED of procaspase-8 to form the so-called death inducing signaling complex (DISC) Studies in cultured cells using the actin inhibitor Ltn A suggested that this step requires actin filaments. In a final step, processed caspase-8 is freed from the DISC and starts to process its substrates such as caspase-3 or the proapoptotic Bcl-2 family member Bid
Fas/Apo1/CD95,作为一类在结合了FASL 之后在有效的细胞凋亡信号中所需要的化合物,当Fasl 结合到了与Fas/Apo1/CD95同源的受体蛋白,一个细胞内部分具有整个受体蛋白将近90个AA 的细胞凋亡保守区域,和一个由两部分构成的受体分子----FADD 在同型上的相互作用,这里快速的对于FADD 的补充来生成这些受体复合物的过程和微聚团的形成有关,FADD 的凋亡功能保守区域DED 蛋白的N 端依次和前体蛋白酶8的DED 作用从而生成所谓的凋亡诱导信号复合物(DISC ),在利用这种肌动蛋白抑制剂LtnA 培养细胞的过程中,证明了这个过程需要肌动蛋白纤维,在最终的阶段中,处理CASPASE-8是不需要DISC 的作用的,并且开始处理像蛋白酶3或者是细胞凋亡前体BCL-2蛋白家族成员Bid
◆ 2. CASPASE-9 ACTIVA TION IN THE APOPTOSOME(CASPASE-9,Apaf-1,Cyto-c)
◆ Cell-free assays have demonstrated that cytochrome c interacts with apoptotic protease activating factor-1 (Apaf-1) to form a
multiprotein complex containing active caspase-9 in the presence of dATP . This complex is also known as the apoptosome. The finding that caspase-9 enzymatic activity increases 1000-fold upon its association with the apoptosome led to the current model whereby the assembly of this scaffolding complex is the key molecular event in the activation of caspase-9. This Apaf-1/caspase-9 holoenzyme has been shown to activate executioner caspases such as caspase-3
无细胞实验已经证明细胞色素C 和细胞凋亡蛋白酶激活因子1*(APAF-1)在dA TP 的存在下相互作用形成了一个包含有有活性的蛋白酶9的多蛋白聚合体,这个复合物也被称为细胞凋亡小体,这个关于细胞凋亡蛋白酶9和细胞凋亡小体结合后酶活力增加1000倍的发现导致了现有的关于这种依靠这种支架型复合物的研究模型成为在激活细胞凋亡蛋白酶9活动中的关键性的分子活动,这种Apaf-1/caspase9全酶已经被证实可以激活像细胞凋亡蛋白酶比如蛋白酶3
Apaf-1 contains at least three functional domains: (细胞蛋白激活因子包括至少三个的功能区域)
(i) an N-terminal CARD (caspase recruitment domain) that is
required for binding caspase-9,
(ii) a domain homologous to Ced-4 which mediates Apaf-1
self-organization (一个与线虫ced-4同源的保守区域,可以调节apaf-1的自我组织)
(iii) 12 or 13 C-terminal WD-40 repeats thought to be involved in
protein-protein interactions
3Highly purified recombinant cytochrome c, Apaf-1 and caspase-9 proteins were demonstrated to form a 1.4 mDa complex
as visualized by gel filtration experiments.
高度纯化的重组细胞色素C,APAF-1和细胞凋亡蛋白酶9被证明是形成一个1.4mDA 的复合物,这种复合物可以通过葡萄糖凝胶电泳实验获得
• In agreement with these findings in cell-free assays, Apaf-1 was reported to be present in high molecular weight complexes in
cell extracts upon treatment of lysates with dATP and incubation at 37°C. 和这些发现可以吻合的是在实验中,细胞凋亡蛋白激活因子1被报道为在37度以及dA TP 条件下进行的细胞溶解实验所获得的细胞提取物 中的一种大分子量的化合物,
• 700 kDa. The smaller complex appears to form more rapidly and has a higher caspase processing activity. (apaf-1的化学结构:是一个寡聚蛋白)
• Apoptosis induced in human tumor cells by etoposide or N-tosylphenylalanylchloromethyl ketone (TPCK) resulted in the
formation of a 700 kDa complex suggesting a functional relevance in vivo(细胞凋亡可以引起人类肿瘤细胞)
4.The molecular mechanisms of caspase-9 activation within the apoptosome:
In gel filtration experiments using recruitment and processing of which was dependent on the on caspase-9. Interestingly, cleavage-resistant caspase-9 mutants can ◆ The three-dimensional structure of the apoptosome has been determined at a resolution of 27A using electron cryomicroscopy.
Assembly of purified Apaf-1, cytochrome c and dA TP resulted in a wheel-like particle of 7-fold symmetry. Known high-resolution domain structures such as the Apaf-1 CARD or WD40 domains of other proteins enabled the determination of the domain architecture by positioning structural elements within the 3D structure. ◆ In healthy cells, a mechanistic model for the assembly of the apoptosome has been proposed where the N-terminal CARD
domain of Apaf-1 is bound to a Y -shaped structure formed by the C-terminal WD40 domains. Cytochrome c displaces the CARD domain and this change in Apaf-1 conformation enables oligimerization of CARD domains to form the wheel-like structure of the apoptosome.
翻译:(划线部分)在对于使用dA TP 激活的细胞溶解物的葡萄糖凝胶电泳实验中,细胞凋亡蛋白酶3在某些细胞凋亡小体中被检测到,这个对于蛋白酶3的补充和处理依靠的是细胞凋亡蛋白酶9,有趣的是,对于分解有抗性的细胞凋亡蛋白酶9变异体仍然可以补充和激活蛋白酶3,最终得到的结论是对于细胞凋亡蛋白酶9的处理在这个过程中即不是充分的也不是必须的 对于细胞凋亡小体的三维结构已经在分辨率为27A 的条件下,由冷冻电镜中得出,对于细胞凋亡蛋白酶激活因子1,细胞色素C 和dA TP 的组装导致了7倍重的具有对称性的轮状颗粒。在已知的高分辨率的结构域像是apaf-1 card以及其他蛋白的结构域WD40通过在三D 范围内增加的一些结构性元素使得结构域的结构得到了确定,
5.Caspase Inhibition
◆ THE VIRAL CASPASE INHIBITORS P35 AND CRMA(滤过性病毒的蛋白酶抑制剂P35和CrmA )
关于p35的介绍
◆ Many viruses have evolved mechanisms to prevent apoptosis of their host cell in order to enable sustained viral replication. ◆ The p35 gene of the baculovirus Autographa california multiply embedded nuclear polyhedrosis virus (AcMNPV) encodes a
potent and broad-acting caspase inhibitor that can inhibit caspase activity and cell death in nematode, insect and mammalian systems. Infection of SF21 cells derived from the Spodoptera frugiperda (Order Lepidoptera) by a p35-deficient baculovirus causes cell death and prevents viral replication. In addition to blocking apoptosis induced by viral infection, p35 can inhib it insect cell death caused by overexpression of activated insect SF-caspase-1 or mammalian caspase-3. Overexpression of p35 can protect mammalian cells from various forms of apoptosis. Expression of p35 in transgenic nematodes, flies and mice produces phenotypes characterized by excessive developmental cell death.
关于crmA 的介绍
• Cowpox virus expresses the protein CrmA (cytokine response modifier A) in order to avoid inflammatory and apoptotic
responses following host cell infection.
• CrmA targets members of the caspase family of proteases that either initiate apoptosis pathways (caspases-8 and -10) or trigger
activation of the pro-inflammatory cytokines interleukin-1 and interleukin-18
• Similar to p35, CrmA inhibits proteases by acting as a pseudosubstrate. (竞争性抑制作用),和p35一样,crmA 通过表现
成为一个假底物来抑制蛋白酶的活性
• However, structural analysis showed that CrmA represents a true member of the serpin family of protease inhibitors. CrmA can
also inhibit the serin protease Granzyme B and therefore represents a cross-class inhibitor of proteases.
Chapter2
1. 名词解释:BCL-2 第一致癌基因,包括促细胞凋亡基因和抗细胞凋亡基因,具体见chapter2的表格
2. 关于Bcl-2家族的分析
(1)Bax
◆ 1) Bax, Bc l-2 associated X protein, was the first of the other family members to be discovered by
co-immunoprecipitation (免疫沉淀反应) studies with Bcl-2.
2)Bax shows a large degree of conservation in its BH1 and BH2 regions with Bcl-2.
3)分子水平的性质:
◆ The finding that the Bax gene promoter contains four p53 binding sites indicates that it is up-regulated at the transcriptional
level by p53 and thus that Bax may function as a primary response gene in the p53-mediated pathway of apoptosis initiation. ◆ Bax expression can be modulated by other factors as its mRNA levels have been shown to be down-regulated in IL-6 treatments
of leukaemia cell lines.
Bax 基因启动子的性质:1. 四个p53结合位点,在p53胞凋亡开始阶段由p53介导的通路中是一个主要的作用基因
的作用
(2)Bcl-xl
◆ Bcl-xL was initially isolated in chicken lymphoid cells (淋巴细胞)using a cDNA Bcl-2 probe and was found to share 44%
sequence homology (同源)with Bcl-2.
◆ It was also shown to interact with other family members in a similar manner to that of Bcl-2.
◆ Bcl-xL exhibits high structural conservation to Bcl-2 in that it contains the essential BH1 and BH2 domains, however the splice
variant form, Bcl-xS only contains the BH3 and BH4 domains.
◆ Both the level and pattern of expression of Bcl-x is different from that of Bcl-2 with expression levels generally higher for Bcl-x
in all tissues with the exception of the lymph nodes, however the subcellular distribution of the two proteins are similar indicating similar functions.
(3)bak
来源:Bak (Bcl-2 homologous antagonist(拮抗物) / killer) was cloned from human heart and Epstein-Barr transformed human B-cells with three closely related Bak genes found on three different chromosomal locations
同源性:contained the same hydrophobic carboxy(疏水羰基)-terminal domain found on Bcl-2 and Bcl-xL
作用:Bak has been shown to promote apoptosis in response to IL-3 withdrawal but inhibits apoptosis in response to serum withdrawal
◆ Bak is shown to co-immunoprecipitate with Bcl-xL when under non-apoptosis inducing conditions but does not
co-immunoprecipitate following apoptotic stimulation.
◆ This suggests that upon stimulation of apoptosis, Bak undergoes a conformational change resulting in dissociation of Bak from
Bcl-xL leaving Bak free to exert its pro-apoptotic effect.
(4)Bad
◆ 来源:Bad (Bcl-xL/ Bcl-2 associated death promoter homologue(同族)) was shown to heterodimerise(异二聚物) with Bcl-2
in vivo(在体内) but was first discovered by its interaction with Bcl-2 in a yeast two-hybrid system(杂交).
◆ 同源性:Bad shares limited sequence homology with Bcl-2 but the functional significant sequences within the BH1 and BH2
domains are conserved.
作用:Bad plays an important role in the dissociation of Bax from its complex with Bcl-2 or Bcl-xL due to its regulation by phosphorylation (磷酸化作用).
(5)MCL-1 human myeloid differentiation protein
◆ 作用:protect cells against constitutively induced apoptosis by expression of Bax or c-myc, however, its anti-apoptotic effect is
not as great as that exhibited by Bcl-2.
在细胞凋亡中保护细胞在结构上免受能够诱导细胞凋亡的基因bax 或者细胞色素c 的作用,但是它的作用并没有Bc l-2表现出来的那么明显
分布情况的讨论:The tissue distribution of Mcl-1 compared to that of Bcl-2 is significantly different indicating that maybe Mcl-1 is an alternative to Bcl-2 where tissues cannot express Bcl-2. 结论:It is theorised (理论化)that Mcl-1 blocks apoptosis until Bcl-2 can be up-regulated.(成理论化的结论是mcl-1将阻止细胞凋亡,直到bcl-2的表达情况升高)
(6)A1
来源:early response gene ,A1 interacts specifically with Bax but not with any other Bcl-2 family members.
作用:TNF-induced apoptosis in the presence of actinomycin (放射菌素)D and inhibit ceramide(神经酰胺) cell death in endothelial cells (内皮细胞).
(7)BID
◆ 作用:a BH3 interacting domain death agonist that was initially identified due to its interaction with both Bcl-2 and
Bax proteins. Bid interacts with both death agonists(拮抗剂) and antagonists(对抗剂) but does not form homodimers. However it was observed by mutational analysis that the BH3 domain was crucial for interaction with Bax and Bcl-2.
◆ 同源性:it only contained homology within the BH3 domain and is predominantly localised within the cytoplasm
with only a small fraction of the cellular levels located in membranes.
(其余因子略)
3.cell engineering approaches of bcl-2 family
The use of the Bcl-2 family in cell engineering is a topic of great interest and can manifest in two main approaches.
1)increasing apoptosis either by decreasing the anti-apoptosis members of the family or increasing the pro-apoptotic members of the family.
2)decrease the likelihood of apoptosis to allow the cell to survive irrespective of(不考虑) the conditions and signals that it faces 用途 :1)作为免疫学的研究目标,具体体现在:对体内免疫细胞B 和T 细胞的保护作用
As Bcl-2 family members maintain a central role in life and death decisions they play a pivotal role in the homeostasis of immune cells at every point where such a decision is required. Whether it is the development of B-cells to secret antibody required, the maintenance of memory cells or the activation induced cell death of T and B cells once their function is completed, it is clear that the malfunction of the Bcl-2 family at these critical times can lead to catastrophic problems within the cell.
使得外部信号只在有限以及特殊的组织中进行,从而保证淋巴球只在特定的位置合成,BCl-2和bcl-xl 可以保证诱导细胞凋亡的正常进行
Extrinsic signals act in a limited and tissue specific manner to ensure the correct numbers of lymphocytes are produced at the correct location. Bcl-2 and Bcl-xL are capable of preventing neglect-induced cell death. Bcl-2 and Bcl-xL transgenic mice accumulate vast amounts of lymphocytes depending on cell type targeted and the increase in numbers is gene-dose dependant, however the number that are produced and the number that survive exhibit large discrepancies indicating that Bcl-2 and Bcl-xL can not be solely responsible.
介绍了A1基因的性质:缺乏A1基因的血细胞如嗜中性粒细胞将会加速其的细胞凋亡
A1, shows great homology to Bcl-2 ,is necessary for haemopoietic (造血) cell survival as its deletion leads to accelerating neutrophil (嗜中性粒细胞)apoptosis. Under infection conditions A1 has been shown to be induced, in order to allow the cells to survive an acute inflammatory(发炎的) response demonstrated by the lack of such a response in A1-deficient cells.
MCL-1的性质:1)加强细胞的生存时间,2)在细胞凋亡过程中增强了和细胞生存有关的mcl-2基因的表达
The myeloid cell leukemia-1 gene (Mcl-1) was first identified by its increased expression early on in the differentiation of a human myeloid leukemic cell line. In normal peripheral blood B cells treated with agents that either promote survival or enhance cell death the up-regulation of Mcl-1 correlates with cell survival and the down-regulation of Mcl-1 correlates with cell death.
2)bcl-2基因家族在癌症治疗中的角色以及将成为在治疗中的潜在利用目标
主要是以下几点
1’ 高水平的bcl-2的表达被认为是人类罹患癌症的重要标志
2’ 对于bcl-2基因的翻译后修饰的认识可以利用到药物学上的应用,提高传统药物的效力。
3/对肿瘤细胞是否起作用取决于小分子阻遏物的作用
4. 基因治疗的利用
实例见课件
The expression level of Bcl-2 also correlates with relative resistance to a spectrum of chemotherapeutic drugs and γ-irradiation,
which thus seem to emerge on the apoptotic pathway at the same point to execute apoptosis.Bcl-2基因的表达水平同样和γ射线以及化学治疗药物的相对抗性有关
Post-translational modifications(翻译后修饰) of Bcl-2, such as phosphorylation and proteolysis can lead to modulation of its protective function. The evidence that drugs targeted to the mitochondria and DNA damaging agents may induce phosphorylation of Bcl-2, suggests the possibility of exploiting novel targets to improve the therapeutic efficacy of conventional drugs and circumvent the Bcl-2 mediated response to apoptosis.
Bcl-2用在基因治疗
Gene therapy is generally used to replace genes that have been lost or whose expression is low resulting in the accumulation of malignancies (恶性). In most cancers it is the antiapoptotic genes that are over-expressed however as mentioned previously the balance between the anti- and pro-apoptotic members provides the decision point of whether a cell should enter apoptosis or not.
利用基因治疗的原理:relies on the increase of pro-apoptotic molecules within the cell to counterbalance (使平衡)the overexpression of the anti-apoptotic molecules.
3’展示bcl-2家族在加强生化产量上的应用
4. Growth Factors, Survival Factors and Apoptosis(IGF)
Cytokines such as stem cell factor (SCF), erythropoietin (EPO), interleukin (IL)-3 and IL-2 promote survival of different
populations of hematopoietic cells and allows them to differentiate or carry out their functions in the immune system.
Neurons are dependent on survival factors such as nerve growth factor (NGF) and other neurotrophins produced by their target tissues to survive and be maintained
IGF 的作用机理:见(chpter 2 3.3)
Chapter3
1. 不同种类的生物细胞凋亡的特征对比
植物细胞细胞凋亡的特征
具体表现在
• Cell shrinkage(细胞收缩)
• chromatin condensation(染色质固缩)
• DNA fragmentation( DNA 断裂)
• Internucleosomal DNA cleavage (核小体DNA 裂解)
• The formation of structures that resemble apoptotic bodies
动物细胞细胞凋亡的特征
• 形态学上的特征包括:nuclear alterations, chromatin condensation and fragmentation of the nucleus, cell shrinkage,
surface alterations such as membrane blebbing, and the formation of membrane bound vesicles termed apoptotic bodies.
2. Necrosis 的特征
• 定义:accidental or passive death of cells caused by a loss of cell integrity (完整性) and compartmentalization of
organelles. (细胞起的划分)
• 特征:Its characterics are as follows∶
a) cell swelling (细胞溶胀)
b) disruption of membranes (膜破裂)
c) metabolic insult(代谢损伤)
3. 细胞凋亡和细胞死亡的区别
4. 热反应和冷反应(见3.1)
5. 两种不同的受体通路:死亡受体通路和线粒体途径
5. 植物荷尔蒙(激素)(6.1)
Chapter4
1. 关于细胞凋亡蛋白酶的阻遏物
caspase( cysteine-containing aspartate-spicific protease) 意为含天冬氨酸特异性半胱氨酸蛋白酶, 是细胞凋亡的主要执行者。在caspase 家族中, caspase-8是重要的启动型caspase, 可活化caspase-3等效应型caspase, 使细胞凋亡。
哺乳动物caspase 的活化主要有细胞外(caspase-8途径/非线粒体途径) 和细胞内( caspase-9途径/线粒体途径) 两条途径, 且这两条途径有交叉。具体有以下五种物质
.1 CrmA
.2 P35
.3 IAP FAMILY
.4 DOMINANT NEGATIVE CASPASES
5 PEPTIDE INHIBITORS
• CrmA (细胞因子反应调节蛋白)是病毒及内源性的Caspase 抑制剂,属于丝氨酸蛋白酶抑制剂,它能抑制Caspase-1 ,
以及Caspase-8 (the key mediator of the extrinsic death receptor pathway)的活性,but possesses a much weaker affinity (亲和力)for the effector caspase-3。
• P35 是一种来源于杆状毒的Caspase 抑制蛋白。P35 has been shown to provide irreversible Inhibition(不可逆抑制) of
caspases -1,-2, -3, -4, -6,-7, -8, and -10 but not caspase-9,and as a result possesses broader specificity for caspases than CrmA and a preference for the downstream effector caspases.
• IAP (凋亡抑制分子) proteinsare classified based on theexistence of a minimum of one BIR.may or may not contain a RING
finger. cIAP1 and cIAP2 contain a CARD domain(Caspase募集区)
IAPs 在两条主要的细胞凋亡途径中都能阻断细胞凋亡,
• 在死亡受体途径中,IAPs 能通过其BIR 区阻断Caspase-3 ,Caspase-7 的活化。研究发现,XIAP 的BIR2 区能够与
Caspase-7 结合有效抑制其活性, 而BIR1 和BIR2间的连接区则能与Caspase-3 及Caspase-7 的活性中心结合竞争性抑制二者的活性;
• 在线粒体途径中,IAPs 通过三种方式抑制凋亡:
①直接与pro-Caspase-9 作用干扰其加工过程;
②IAPs 的CARD 区与Apaf-1 竞争性结合, 阻断Caspase 活化;
③直接抑制活化的Caspase 。
其余抑制因子的介绍见chapter4.3
2. 工业相关的细胞凋亡抑制剂方法
1)Chemical inhibitors—z-V AD-fmk
2)Concentration:
3)Genetic engineering---Bcl-2
4)Additive---Butyrate(丁酸盐)
利用丁酸盐的原因:
enhancement of protein expression despite its growth inhibitory effect
对蛋白表达的加强,尽管还有一些对于生长的负面影响
5)Protein inhibitor:IAPs ; crmA ; p35
3. Apoptosis pathways的分类
Intrinsic---mitochondrial mediated pathway由caspase-9介导
Extrinsic---a receptor-mediated pathway,caspase-8介导
4. 关于细胞凋亡蛋白酶的介绍:
members of a family of intracellular proteins involved in the initiation and execution of apoptosis. Caspase activation is central to the apoptotic pathway. In recent years the study of the regulation of caspase activation in various cell lines has revealed highly complex mechanisms regulating the fine balance between cell survival and cell death.
caspase-9 may be a central regulator of downstream caspase activation in the cytochrome c dependent pathway.
Caspase-9 dominant negative / caspase 9DN
(caspase-9的显性负调控) 的原理
It is a catalytically inactive caspase-9. Overexpression of caspase-9DN was found to effectively inhibit cell death initiated by a wide variety of inducers, such as FADD, TRADD as well as Ced-4, the C. elegans homologue of Apaf-1 .
The mechanism of inhibition of endogenous caspase-9 is thought to be caused by competition with caspase-9 for binding to Apaf-1 and/or directly by a formation of an inactive heterodimer(异源二聚体) between caspase-9 and caspase-9DN.
5. 细胞凋亡相关的assays (chapter4.4)
磷脂酰丝氨酸外翻分析(Annexin V法):
关于外翻现象的描述:In these cells the membrane phospholipid phosphatidyl serine (PS,磷脂酰丝氨酸) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment.
关于该分析法的描述:a Ca2+ dependent binding protein that binds to PS with a strong affinity. Annexin can be conjugated to fluorochromes such as FITC. In conjunction with a vital dye, propidium iodide, that labels cells with lost membrane integrity, this assay can distinguish cells in early stages of apoptosis (annexin positive, PI negative) from cells in late stage of apoptosis (annexin positive, PI positive).
2)DNA fragmentation
DNA 片段化检测
关于DNA 片段化出现的原因:One of the later stages of apoptosis is the activation of endonucleases (胞内核酸酶),which
translocate(易位) into the nucleus and cause DNA fragmentation and inter-nucleosomal (核小体之间)cleavage. These nucleases degrade the higher order chromatin structures into fragments of 50-300 kbp and subsequently into smaller DNA pieces of about 200bp in length, forming a characteristic “ladder” of DNA fragments which can be detected by agarose gel electrophoresis(琼脂糖凝胶电泳).
检测方法的原理描述:这是一种定量的方法,
DNA breaks can be labeled at the 3’-hydroxyl termini with, e.g., fluorescent(荧光的) tagged deoxyuridine triphosphate(脱氧尿苷三磷酸) nucleotides (FITC-dUTP) in a reaction catalyzed by deoxynucleotidyl transferase (TdT, 脱氧核苷酸(基) 转移酶) and subsequently(随后) analyzed by flow cytometry(流式细胞术)
技术定义:Caspases are cysteine-containing Asp specific proteases (Steinnicke et al., 1997) that are synthesized as inactive precursors(失活前体) and are processed by proteolytic cleavage during apoptosis. Caspase-3 has been implicated as a key executioner caspase and its activation is often used as a measure of apoptosis either directly by assaying caspase-3 activity or indirectly by detecting its proteolytically cleaved endogenous substrates (such as PARP).
技术原理的介绍:The direct measure of caspase-3 activity is based on the utilization of a fluorogenic substrate (Ac- DEVD-AMC), which is a synthetic tetrapeptide that is recognized by caspase-3 and cleaved between D and AMC, releasing the fluorescent AMC that can be quantified by flow cytometry(流式细胞仪) or spectrofluorometry(荧光分光光度法
6.RNAi 干扰技术的介绍:包括snRNAi 和shRNAi, 选择片段的方法见chapter4.6的课件
Chapter5.
1,糖基化和糖基化工程的概念(见名词解释),
2. 糖基化工程的作用(见chapter5.4)
3. 研究糖蛋白的主要技术(具体解释见chapter5.1):
HPLC
• HPAEC
• SDS-PAGE
• CE
• MS
• NMR
4. 糖基化工程的主要途径
1)、人工体外修饰合成
利用已获取的各种糖基转移酶、糖苷酶等,在一定条件下剪接天然的糖链。这种方式具有很强的底物专一性,对修饰目标寡糖链上有限的糖残基是一种方便有效的方法。缺点是剪接工具在种类和功能方面不足,技术手段不很理想,现用来大量制备有一定疗效的药用糖蛋白产物还有许多困难。
2)、利用基因重组方式生产所需的糖复合物
例如, CHO 和BHK ,通常不表达α2,6ST 活性,通过基因工程手段使得α2,6ST 基因进行表达,并导致生产细胞蛋白含有唾液酸残基。对这个实验进行扩展,使得过度表达几种个体的或复合外源性糖基转移酶 ,成功地增加了重组蛋白质中唾液酸和半乳糖含量 ,并引入显著量的末端α2 , 6唾液酸到重组蛋白产物的N 联聚糖中。
5. 天冬酰胺的修饰工程
6. 哺乳动物糖基化的因素 《见chapter5.8 细胞培养过程中蛋白质糖基化的影响因素》