本文選題：SP + NK92-MI��； 參考：《中國醫(yī)科大學(xué)》2009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)時分泌的一種生物活性多肽,廣泛分布于細(xì)的初級傳入神經(jīng)纖維內(nèi)。軀體和精神刺激作用于神經(jīng)系統(tǒng)時,既可通過神經(jīng)的直接作用影響免疫系統(tǒng)的功能活動,也可通過釋放內(nèi)分泌激素或其它細(xì)胞因子對免疫功能進(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)。 前期的研究發(fā)現(xiàn)SP的生物活性是通過與SP受體發(fā)生作用而實現(xiàn)的。許多免疫細(xì)胞膜上存在有SP的特異性受體。這些形態(tài)學(xué)資料為SP參與免疫調(diào)節(jié)提供了證據(jù)。一些免疫細(xì)胞也能夠產(chǎn)生SP,并以自分泌或/和旁分泌的方式調(diào)節(jié)免疫細(xì)胞的功能。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)胞表面有多種活化性受體,但是有關(guān)SP調(diào)節(jié)NK細(xì)胞功能是通過何種受體途徑報道較少。為此,我們檢測了SP對NK-1受體表達(dá)變化的影響以及NK-1受體阻斷劑作用后的NK細(xì)胞增殖和殺傷活性的變化,以研究SP影響NK細(xì)胞功能的可能作用機(jī)制。 材料和方法 1、細(xì)胞培養(yǎng)和處理,收集對數(shù)期細(xì)胞,實驗組加NK-1受體阻斷劑預(yù)處理后與對照組分別加不同濃度P物質(zhì)培養(yǎng)NK92-MI細(xì)胞24h; 2、MTT法檢測NK-1受體阻斷劑作用前后不同濃度SP(10~(-14)mol/L,10~(-12)mol/L,10~(-10)mol/L,10~(-8)Smol/L,10~(-6)mol/L,10~(-4)mol/L)對NK92-MI細(xì)胞增殖的影響; 3、MTT釋放法測定NK-1受體阻斷劑作用前后不同濃度P物質(zhì)對NK92-MI細(xì)胞殺傷活性的影響; 4、RT-PCR檢測比較NK-1受體和NK-2受體mRNA表達(dá)水平。 5、流式細(xì)胞術(shù)檢測比較NK92-MI細(xì)胞表面SP受體的膜表達(dá)情況; 結(jié)果 1、當(dāng)濃度為10-10M、10-8M、10-6M時,SP對NK92-MI細(xì)胞增殖有明顯促進(jìn)作用(P＜0.05)。10~(-10)M時SP對NK92-MI細(xì)胞增殖促進(jìn)作用更為顯著,(P＜0.01)。10~(-7)M濃度的NK-1受體阻斷劑能完全阻斷10~(-10)M、10~(-8)M、10~(-6)M濃度的SP對NK92MI細(xì)胞的增殖促進(jìn)作用。 2、效靶比為4:1時,10~(-14)M～10~(-8)M濃度的SP對NK92-MI細(xì)胞殺傷活性有增強(qiáng)作用;低濃度10~(-14)M較高濃度10~(-8)M殺傷作用明顯。10~(-7)M的NK-1受體阻斷劑能部分阻斷SP對NK92-MI細(xì)胞殺傷活性的增強(qiáng)作用,但是與對照組比較差異無顯著性。 3、RT-PCR方法檢測到NK92-MI細(xì)胞中有NK1受體mRNA的表達(dá),且10~(-10)M,10~(-8)M,10~(-6)M濃度的SP使NK92-MI細(xì)胞中NK1受體mRNA的表達(dá)增強(qiáng),與對照組比較有顯著性差異(P＜0.05);RT-PCR方法未檢測到NK92-MI細(xì)胞中有NK2受體mRNA的表達(dá)。 4、SP與NK92-MI細(xì)胞共孵育24 h,FCAS檢測結(jié)果表明,NK-1受體在10~(-10)M～10~(-6)M濃度SP作用下表達(dá)增強(qiáng)。 結(jié)論 NK92-MI細(xì)胞上有NK-1受體的表達(dá),且一定濃度SP能誘導(dǎo)其表達(dá)上調(diào)。一定濃度范圍的SP對NK92-MI細(xì)胞的增殖有促進(jìn)作用,其中10~(-10)M濃度時促進(jìn)增殖作用最強(qiáng);應(yīng)用NK-1受體阻斷劑可以完全阻斷一定濃度范圍的SP對NK92-MI細(xì)胞的促增殖作用。一定濃度的SP能夠增強(qiáng)NK92-MI細(xì)胞的殺傷活性。10~(-14)M、10~(-12)M、10~(-10)M濃度的SP能夠明顯增強(qiáng)NK92-MI細(xì)胞殺傷作用。應(yīng)用NK-1受體阻斷劑能部分阻斷SP對NK92-MI細(xì)胞的殺傷活性的增強(qiáng)作用.。SP對NK92-MI細(xì)胞的促增殖作用是由NK-1受體介導(dǎo)的;而SP對NK92-MI細(xì)胞殺傷活性的影響可能有部分是通過NK-1受體以外途徑發(fā)揮作用的。
[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 .


NK cells are a kind of important lymphocyte in innate immune system . NK cells are a kind of very important lymphocyte in innate immune system . NK cells are a kind of very important lymphocyte in innate immune system . NK cells play an important role in resisting infection , anti - tumor , immunoregulation and hematopoietic system . NK cells can regulate NK - 1 receptor expression change and NK cell proliferation and kill activity after the action of NK - 1 receptor blocking agent to study the possible mechanism of SP on NK cell function .


Materials and Methods


1 . Cell culture and treatment were performed to collect logarithmic phase cells . After pretreatment with NK - 1 receptor blocking agent , NK92 - MI cells were cultured with different concentrations of substance P in the control group .


2 . The effects of different concentrations of SP ( 10 - 14 ) mol / L , 10 - 12 mol / L , 10 - 10 mol / L , 10 - 8 mol / L , 10 - 6 mol / L , 10 - 4 mol / L on the proliferation of NK92 - MI cells were detected by MTT assay .


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


4 . The expression level of NK - 1 receptor and NK - 2 receptor mRNA was detected by RT - PCR .


5 . Flow cytometry was used to detect the expression of SP receptor on the surface of NK92 - MI cells .


Results


1 . When the concentration was 10 - 10M , 10 - 8M , and 10 - 6M , the proliferation of NK92 - MI cells was significantly enhanced by SP ( P < 0 . 05 ) .


2 . The cytotoxicity of SP on NK92 - MI cells was potentiated by SP of 10 ~ ( -14 ) M ~ 10 ~ ( -8 ) M at 4 : 1 , and the cytotoxicity of SP on NK92 - MI cells was markedly blocked by 10 ~ ( -7 ) M NK - 1 receptor blocking agent , but there was no significant difference compared with the control group .


3 . The expression of NK1 receptor mRNA in NK92 - MI cells was detected by RT - PCR , and the expression of NK1 receptor mRNA in NK92 - MI cells was significantly higher than that in control group ( P < 0.05 ) .


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本文編號：1989904