本文選題：物質(zhì) + NK92MI�。� 參考：《中國醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 前言 P物質(zhì)(Substance P,SP)是神經(jīng)內(nèi)分泌系統(tǒng)反應(yīng)時(shí)分泌的一種生物活性多肽,廣泛分布于細(xì)的初級(jí)傳入神經(jīng)纖維內(nèi)。軀體和精神刺激作用于神經(jīng)系統(tǒng)時(shí),既可通過神經(jīng)的直接作用影響免疫系統(tǒng)的功能活動(dòng),也可通過釋放內(nèi)分泌激素或其它細(xì)胞因子對(duì)免疫功能進(jìn)行調(diào)節(jié),而經(jīng)典神經(jīng)遞質(zhì)、神經(jīng)肽則是直接作用于免疫活性細(xì)胞和其他參與免疫反應(yīng)細(xì)胞的重要調(diào)節(jié)物質(zhì)。SP的生物活性是通過與速激肽受體(NK-R)的亞型NK-1(neuorkinin-1),NK-2,NK-3的結(jié)合表現(xiàn)出來的。 SP與臨床的多種疾病有關(guān),SP在不同條件下所引起的作用具有復(fù)雜性和多樣性。進(jìn)一步研究SP在神經(jīng)內(nèi)分泌-免疫網(wǎng)絡(luò)中的具體作用機(jī)制會(huì)對(duì)炎性疾病及免疫性疾病的病理生理本質(zhì)有更新的認(rèn)識(shí),以期對(duì)臨床防治有所幫助。 NK細(xì)胞是天然免疫系統(tǒng)中一類十分重要的淋巴細(xì)胞,在機(jī)體抗感染、抗腫瘤、免疫調(diào)節(jié)及造血系統(tǒng)等方面發(fā)揮重要作用。NK細(xì)胞通過細(xì)胞介導(dǎo)的細(xì)胞毒殺傷非己細(xì)胞,并通過釋放多種細(xì)胞因子(如IFN,GM-CSF和TNF)和趨化因子調(diào)節(jié)天然免疫和獲得性免疫。NK細(xì)胞的殺傷活性由其抑制性受體和活化受體調(diào)控。 SP在體內(nèi)體外均可調(diào)節(jié)多種免疫功能,但SP對(duì)NK細(xì)胞功能影響的報(bào)道較少。為此,我們檢測(cè)了SP是否可以促進(jìn)NK細(xì)胞增殖和殺傷活性,并測(cè)定SP對(duì)NKG2A/D表達(dá)的影響,以研究SP對(duì)NK細(xì)胞功能的影響及可能的作用機(jī)制。 材料和方法 1、細(xì)胞培養(yǎng)和處理,收集對(duì)數(shù)期細(xì)胞加不同濃度P物質(zhì)培養(yǎng)24h; 2、MTT法檢測(cè)不同濃度SP(10~(-6)mol/L,10~(-8)mol/L,10~(-9)mol/L,10~(-10)mol/L,10~(-12)mol/L,10~(-14)mol/L)對(duì)NK92-MI細(xì)胞增殖和細(xì)胞周期的影響; 3、MTT釋放法測(cè)定不同濃度P物質(zhì)對(duì)NK92-MI細(xì)胞殺傷活性的影響; 4、流式細(xì)胞術(shù)檢測(cè)比較NK92-MI細(xì)胞抑制性受體NKG2A和活化性受體NKG2D的膜表達(dá)情況; 5、RT-PCR檢測(cè)比較NKG2A和NKG2D的mRNA表達(dá)水平。 結(jié)果 1、10~(-6)M-10~(-10)M濃度的SP對(duì)NK92-MI細(xì)胞的增殖和活力有促進(jìn)作用,與對(duì)照組比較有顯著性差異。 2、SP在10~(-6)mol/L,10~(-8)mol/L,10~(-14)mol/L濃度時(shí),作用24h后,實(shí)驗(yàn)組的DNA合成前期細(xì)胞所占百分比(G1%)下降,而DNA合成期細(xì)胞所占百分比(S%)增高,反應(yīng)增殖活力的增殖指數(shù)PrI值有明顯增加:10~(-4)mol/L的SP使S期細(xì)胞比例下調(diào),G1期百分比增加。 3、效靶比為4:1時(shí),各濃度SP;NNK細(xì)胞殺傷活性顯示有增強(qiáng)作用,此作用強(qiáng)度隨SP濃度的增加而降低。效靶比1:1時(shí),SP對(duì)NK92-MI細(xì)胞殺傷活性的影響不明顯。 4、SP與NK92-MI細(xì)胞共孵育24h,FCAS檢測(cè)結(jié)果表明,NKG2A在10~(-8)-10~(-12)M濃度SP作用下NKG2A表達(dá)均有上調(diào),當(dāng)SP在10~(-14)M濃度時(shí),NKG2A的表達(dá)下調(diào),但無統(tǒng)計(jì)學(xué)意義。NKG2D各組SP濃度較對(duì)照組均有不同程度上調(diào)。 5、各濃度SP與NK92-MI細(xì)胞共孵育24h,采用RT-PCR方法對(duì)部分功能受體mRNA表達(dá)變化情況進(jìn)行檢測(cè),結(jié)果顯示NKG2A各濃度SP組較對(duì)照組mRNA均有不同程度增強(qiáng);各濃度組NKG2D的mRNA表達(dá)均有上調(diào)。 結(jié)論 一定范圍濃度的SP對(duì)NK92MI細(xì)胞的增殖有促進(jìn)影響(存在劑量依賴性)。一定濃度的SP能夠增強(qiáng)NK92-MI細(xì)胞的殺傷活性,各濃度SP均上調(diào)NKG2D的膜表達(dá)水平和mRNA水平(兩者變化趨勢(shì)完全一致),上調(diào)作用隨濃度增大而增強(qiáng)。10~(-8)M、10~(-10)M和10~(-12)M組NKG2A膜表達(dá)和mRNA水平均有上調(diào),10~(-6)M組mRNA水平上調(diào)而膜表達(dá)無明顯變化。低濃度的SP(10~(-14)M)不能上調(diào)NKG2A的膜表達(dá),而NKG2D表達(dá)上調(diào),可能為此濃度SP促殺傷活性顯著增強(qiáng)的原因之一。
[Abstract]:Foreword


Substance P ( SP ) is a kind of bioactive polypeptide secreted by neuroendocrine system . It is widely distributed in the primary afferent nerve fiber . The body and the spirit stimulate the function of the immune system through the direct action of nerve , but the classical neurotransmitters and neuropeptides act directly on immune active cells and other important regulating substances involved in the immune response cells . The biological activity of SP is demonstrated by the combination of the subtypes of NK - 1 , NK - 2 , NK - 3 with tachykinin receptor ( NK - R ) .


SP is associated with a variety of clinical diseases , and SP plays a role in complexity and diversity under different conditions . Further research on the specific mechanism of SP in neuroendocrine - immune network can be helpful for clinical prevention and treatment .


NK cells are a very important lymphocyte in innate immune system . NK cells play an important role in anti - infection , anti - tumor , immunoregulation and hematopoietic system . NK cells can kill non - hexyl cells through cell - mediated cytotoxicity and regulate innate immunity and acquired immunity by releasing various cytokines ( such as IFN , GM - CSF and TNF ) and chemokine . The killing activity of NK cells is regulated by inhibitory receptors and activated receptors .


The effects of SP on NK cell proliferation and killing activity were detected , and the effects of SP on NKG2A / D expression were measured to investigate the effects of SP on NK cell function and possible mechanism .


Materials and Methods


1 , cell culture and treatment , collection of logarithmic phase cells plus different concentration of substance P for 24h ;


2 . The effects of different concentrations of SP ( 10 ~ ( -6 ) mol / L , 10 ~ ( -8 ) mol / L , 10 ~ ( -9 ) mol / L , 10 ~ ( -10 ) mol / L , 10 ~ ( -12 ) mol / L , 10 ~ ( -14 ) mol / L ) on the proliferation and cell cycle of NK92 - MI cells were determined by MTT assay .


3 . The effects of different concentrations of substance P on the cytotoxicity of NK92 - MI cells were determined by MTT assay .


4 . Flow cytometry was used to detect the expression of NKG2A and NKG2D in NKG2A and NKG2D .


5 . The mRNA expression level of NKG2A and NKG2D was detected by RT - PCR .


Results


The effects of SP on the proliferation and viability of NK92 - MI cells in the concentration of 1 , 10 ~ ( -6 ) M - 10 ~ ( -10 ) M were significantly different from those in the control group .


After 24h , the percentage of DNA synthesis ( G1 % ) in the experimental group decreased , while the percentage of the cells in the experimental group ( S % ) increased , and the proliferation index PrI of the cells increased significantly : 10 - ( -4 ) mol / L SP decreased the proportion of S - phase cells and the percentage of G1 phase increased .


3 . When the target ratio was 4 鈭，

本文編號(hào)：1869478