陳劍虹 王小璞 農(nóng)海寧 鮑 晉 盧宜民

廣州中醫(yī)藥大學(xué)祈福醫(yī)院, 廣東 廣州 511495

深部熱療對(duì)AD離體細(xì)胞治療模式的探索及療效

陳劍虹 王小璞 農(nóng)海寧 鮑 晉 盧宜民*

廣州中醫(yī)藥大學(xué)祈福醫(yī)院, 廣東 廣州 511495

目的：探索深部熱療對(duì)AD離體細(xì)胞的最佳治療模式并評(píng)估治療效果。方法：制備離體AD細(xì)胞模型，隨機(jī)分成對(duì)照組、對(duì)照組熱療組、AD模型組、AD模型熱療組，分別置于41.5℃加熱30、45、60、75、90min，于600nm處測(cè)吸光度(A)值，計(jì)算最佳熱療時(shí)間。造模成AD細(xì)胞后置于41.5℃加熱60min，加熱后繼續(xù)置于37℃恒溫孵育24h，分別于加熱后2、4、8、12、24、72h取出，使用流式細(xì)胞儀行活細(xì)胞膜HSP70表達(dá)量，計(jì)算最佳熱療間隔時(shí)間。4組細(xì)胞熱療后分別加入20μmol/L的Aβ25-35溶液培養(yǎng)24h，應(yīng)用流式細(xì)胞儀檢測(cè)細(xì)胞活力，評(píng)估熱療治療AD效果。結(jié)果：對(duì)照組和AD模型組分別給予不同時(shí)間41.5℃熱療，兩組細(xì)胞從加熱60min開始細(xì)胞增殖趨勢(shì)與非加熱細(xì)胞相比開始下降(P<0.05)，這種下降趨勢(shì)在75min達(dá)到最大，確定細(xì)胞加熱的安全有效時(shí)間為60min，同時(shí)對(duì)照熱療組與AD模型熱療組對(duì)比，兩組細(xì)胞隨溫度變化的細(xì)胞增殖趨勢(shì)基本相同，兩者比較無(wú)統(tǒng)計(jì)學(xué)差異(P>0.05)，表明兩組細(xì)胞的最佳熱療時(shí)間均為60min。AD模型組細(xì)胞在熱療后2、4、8、12、24及72h各時(shí)間段監(jiān)測(cè)的HSP70表達(dá)量均不同，AD細(xì)胞在24hHSP70表達(dá)量均達(dá)到最大值(P<0.05)，其后HSP70表達(dá)量開始下降，表明24h是最佳的熱療間隔時(shí)間。4組細(xì)胞與Aβ作用后加熱，MTT檢測(cè)表明4組細(xì)胞活力呈溫度依賴性上升趨勢(shì)(P<0.05)，表明熱療可有效的減少Aβ的聚集，促進(jìn)Aβ的清除，保護(hù)AD細(xì)胞。結(jié)論：深部熱療對(duì)AD離體細(xì)胞的最佳熱療時(shí)間為60min，最佳熱療間隔時(shí)間為24h，深部熱療可以有效的清除Aβ，促進(jìn)神經(jīng)元細(xì)胞修復(fù)。

深部熱療; AD; 離體細(xì)胞; 治療模式

阿爾茨海默病(Alzheimer Disease，AD)，是一種中樞神經(jīng)系統(tǒng)變性病，起病隱匿，以進(jìn)行性記憶力障礙、認(rèn)知障礙和精神異常為主要臨床特征的一種老年癡呆性疾病[1]。AD病理特征是神經(jīng)元細(xì)胞外液β蛋白(β-amyloid，Aβ)沉積形成神經(jīng)炎性斑，神經(jīng)元內(nèi)神經(jīng)纖維沉結(jié)以及海馬皮層區(qū)域神經(jīng)元的大量丟失[2]。近年來(lái)大量研究發(fā)現(xiàn)，AD患者顱腦內(nèi)神經(jīng)元中熱休克蛋白(Heat Shock Protein，HSP)表達(dá)較正常人明顯減少。神經(jīng)元細(xì)胞在加熱或其他損傷性刺激時(shí)會(huì)產(chǎn)生保護(hù)性的HSP，其中HSP70是中樞神經(jīng)系統(tǒng)含量最豐富的HSP，其具有抑制Aβ聚集，從而保護(hù)神經(jīng)元的作用[3]。本實(shí)驗(yàn)通過(guò)給P12神經(jīng)細(xì)胞株注射Aβ25-35建立AD細(xì)胞模型，觀察不同熱療時(shí)間，不同熱療間隔期對(duì)AD細(xì)胞的活性及HSP70的表達(dá)，探索深部熱療對(duì)AD細(xì)胞的最佳治療模式并評(píng)估治療效果。

1 材料與方法

1.1 材料 Aβ25-35、二甲基亞礬(DMSO)和四甲基偶氮唑鹽(MTT)均為美國(guó)Sigma公司產(chǎn)品；其余均為國(guó)產(chǎn)分析純?cè)噭?。儀器：倒置相差顯微鏡由日本Olympus公司購(gòu)進(jìn)；恒溫CO2培養(yǎng)箱為美國(guó)Thermo公司；FACSCalibur流式細(xì)胞儀由美國(guó)BectonDicknson公司購(gòu)進(jìn)；多功能酶標(biāo)儀由德國(guó)BMG公司購(gòu)進(jìn)。

1.2 試驗(yàn)方法

1.2.1 觀察在離體細(xì)胞水平 觀察熱療后HSP70的表達(dá)，摸索安全的熱療加熱時(shí)間及最佳的熱療間隔時(shí)間。

1.2.1.1 制備離體AD細(xì)胞模型 給予20μmol/L Aβ25-35孵育神經(jīng)細(xì)胞株P(guān)12細(xì)胞(由暨南大學(xué)提供)培養(yǎng)24h。

1.2.1.2 選擇合適的熱療時(shí)間 取對(duì)數(shù)生長(zhǎng)期P12細(xì)胞，以1×104個(gè)細(xì)胞，100uL/孔接種于96孔培養(yǎng)板，繼續(xù)于孵育箱中培養(yǎng)24h，隨機(jī)分成對(duì)照組、對(duì)照組熱療組、AD模型組、AD模型熱療組，分別置于41.5℃加熱30、45、60、75、90min，每個(gè)時(shí)間點(diǎn)各組設(shè)5個(gè)復(fù)孔，實(shí)驗(yàn)結(jié)束后，每孔加5mg/mL MTT0.015mL，置CO2培養(yǎng)箱內(nèi)培養(yǎng)4h，棄上清，每孔加0.15mL DMSO，充分震蕩后，于600nm處測(cè)吸光度(A)值。

1.2.1.3 選擇合適的熱療間隔時(shí)間 熱效應(yīng)存在熱耐受性，現(xiàn)根據(jù)HSP70的表達(dá)量變化明確最佳熱療間隔時(shí)間。取對(duì)數(shù)生長(zhǎng)期P12細(xì)胞，以1×104個(gè)細(xì)胞，100ul/孔接種于96孔培養(yǎng)板，繼續(xù)于孵育箱中培養(yǎng)24h，造模成AD細(xì)胞后置于41.5℃加熱Xmin(根據(jù)1.2.1.2摸索出的時(shí)間)，加熱后繼續(xù)置于37℃恒溫孵育24h，分別于加熱后2、4、8、12、24、72h取出，加熒光標(biāo)記的抗HSP70mAb，使用流式細(xì)胞儀行活細(xì)胞膜HSP70表達(dá)量，根據(jù)HSP70表達(dá)量的變化趨勢(shì)避開AD細(xì)胞的熱耐受期，選擇最佳的熱療間隔時(shí)間(Y h)。

1.2.2 標(biāo)準(zhǔn)熱療后離體細(xì)胞水平下觀察各組細(xì)胞的細(xì)胞活力 進(jìn)一步研究熱療在治療AD中的可能作用。①隨機(jī)分成4組，分別為P12對(duì)照組、P12對(duì)照組+熱療組(熱療Xmin，間隔Yh再次給予熱療1次)、AD模型組、AD模型組+熱療組(造模后給藥熱療Xmin，間隔Yh再次給予熱療1次)并且末次熱療后更換培養(yǎng)基，繼續(xù)培養(yǎng)24h。②Aβ25-35溶解后配成0.5mg/mL溶液，37℃、5%CO2培養(yǎng)箱培養(yǎng)，-20℃保存?zhèn)溆茫褂们跋♂?、過(guò)濾。③4組細(xì)胞內(nèi)加入20μmol/L的Aβ25-35溶液培養(yǎng)24h后給予5g/LMTT20μl孵育4h，棄上清加100μL二甲基亞砜(DMSO)，在酶聯(lián)免疫檢測(cè)儀570nm波長(zhǎng)下測(cè)定各孔光吸收值，并將其與正常對(duì)照組吸光度的比值作為相對(duì)細(xì)胞活力。④流式細(xì)胞儀測(cè)定細(xì)胞凋亡：應(yīng)用AnnexinV-PI雙染色檢測(cè)細(xì)胞凋亡。細(xì)胞按1×105/孔的密度接種在6孔板中，給予Aβ25-35干預(yù)6h后用PBS洗滌2次，室溫避光反應(yīng)15min，應(yīng)用流式細(xì)胞儀檢測(cè)。

2 結(jié)果

2.1 不同熱療時(shí)間對(duì)各組細(xì)胞活力的影響 對(duì)照組和AD模型組分別給予不同時(shí)間41.5℃熱療，兩組細(xì)胞在加熱初期(30min和45min)，細(xì)胞增殖趨勢(shì)分別與其對(duì)應(yīng)的非加熱細(xì)胞相比相差不大；從60min開始加熱細(xì)胞增殖趨勢(shì)與非加熱細(xì)胞相比開始下降(P<0.05)，這種下降趨勢(shì)在75min達(dá)到最大，即此時(shí)在兩種培養(yǎng)條件下細(xì)胞的平均A值差距最大，確定細(xì)胞加熱的安全有效時(shí)間為60min，見(jiàn)圖1。對(duì)照熱療組與AD模型熱療組對(duì)比，兩組細(xì)胞隨溫度變化的細(xì)胞增殖趨勢(shì)基本相同，兩者比較無(wú)明顯統(tǒng)計(jì)學(xué)差異(P>0.05)，表明兩組細(xì)胞的最佳熱療時(shí)間均為60min。見(jiàn)圖2。

2.2 不同熱療時(shí)間對(duì)AD細(xì)胞HSP70表達(dá)的影響 AD模型組細(xì)胞在熱療后2、4、8、12、24及72h各時(shí)間段監(jiān)測(cè)的HSP70表達(dá)量均不同，AD細(xì)胞在24hHSP70表達(dá)量均達(dá)到最大值(P<0.05)，其后HSP70表達(dá)量開始下降，表明24h是最佳的熱療間隔時(shí)間。見(jiàn)圖3。

2.3 深部熱療對(duì)各組細(xì)胞存活率的影響 MTT檢測(cè)結(jié)果顯示，Aβ對(duì)兩組細(xì)胞的細(xì)胞毒性與溫度相關(guān)，熱療使溫度升高后作用于P12細(xì)胞或AD細(xì)胞，使細(xì)胞活力呈溫度依賴性上升趨勢(shì)(P<0.05)，高溫對(duì)細(xì)胞活力有明顯提升作用，表明熱療可有效的減少Aβ的聚集，促進(jìn)Aβ的清除，保護(hù)AD細(xì)胞，促進(jìn)神經(jīng)元細(xì)胞修復(fù)。見(jiàn)表1。

組別樣本量吸光度值存活率/%P12對(duì)照組50.741±0.01480.27±4.45P12對(duì)照組+熱療組50.643±0.022*87.46±2.21AD模型組50.736±0.00958.41±3.53AD模型組+熱療組50.619±0.017*72.02±4.18

注：與非熱療組比較，*P<0.05。

3 討論

AD是老年癡呆中最常見(jiàn)的一種類型，其臨床特點(diǎn)為記憶障礙、認(rèn)知障礙和精神異常等，病情漸進(jìn)性加重，嚴(yán)重影響生活與社交，其患病率隨年齡增高而增高，在65歲以上人群中約為5%，85歲以上人群中約20%，其中印度、中國(guó)等亞洲發(fā)展中國(guó)家患病率增加最為明顯[4]。

3.1 深部熱療對(duì)熱休克蛋白的影響 熱休克蛋白(Heat Shock Proteins, HSPs)是指各種應(yīng)激刺激時(shí)細(xì)胞新合成或合成增加的一類蛋白質(zhì)，它作為體內(nèi)重要的分子伴侶之一，參與蛋白質(zhì)的合成、折疊、裝配、轉(zhuǎn)運(yùn)和降解等過(guò)程，以維持細(xì)胞蛋白自穩(wěn)，提高細(xì)胞對(duì)應(yīng)激原的耐受性，并有助于細(xì)胞恢復(fù)正常的結(jié)構(gòu)和機(jī)能[5]。深部熱療是利用高溫產(chǎn)生熱刺激作用在AD患者神經(jīng)中樞，神經(jīng)細(xì)胞在熱刺激反應(yīng)后，會(huì)發(fā)生蛋白質(zhì)圖譜的改變，表現(xiàn)為正常蛋白合成被抑制，應(yīng)激蛋白HSP合成增加，從而達(dá)到減少Aβ聚集，減少寡聚體的形成，減輕神經(jīng)元細(xì)胞的損傷[6-7]。近年來(lái)深部熱療作為一種無(wú)創(chuàng)、無(wú)痛苦、治療費(fèi)用相對(duì)低廉、無(wú)明顯毒副作用的治療方法，臨床廣泛應(yīng)用于惡性腫瘤和炎癥性疾病，并取得較好的臨床收益。大量的基礎(chǔ)研究表明，能夠產(chǎn)生熱休克蛋白的深部熱療在AD患者的臨床治療上具備潛力，但缺乏系統(tǒng)規(guī)范的治療模式的探討及治療效果的評(píng)估[8]。本研究在離體細(xì)胞水平，在加熱的不同時(shí)間，通過(guò)MTT法測(cè)定細(xì)胞活力，AD細(xì)胞在加熱初期(30min和45min)，細(xì)胞增殖趨勢(shì)分別與其對(duì)應(yīng)的非加熱細(xì)胞相比相差不大；從60min開始加熱細(xì)胞增殖趨勢(shì)與非加熱細(xì)胞相比開始下降(P<0.05)，表明AD細(xì)胞熱療的最佳時(shí)間為60min。此后AD模型組在加熱60min后觀察AD細(xì)胞在加熱后不通時(shí)間段的HSP的表達(dá)，結(jié)果表明AD細(xì)胞在24hHSP70表達(dá)量均達(dá)到最大值(P<0.05)，說(shuō)明AD細(xì)胞體外加熱的最佳時(shí)間間隔為24h。

3.2 深部熱療對(duì)Aβ釋放的作用 Aβ是淀粉樣蛋白前體蛋白(Amyloid Precursor Protein, APP)在人體代謝的正常產(chǎn)物之一，神經(jīng)細(xì)胞，尤其是神經(jīng)膠質(zhì)細(xì)胞產(chǎn)生Aβ后，向細(xì)胞外釋放，進(jìn)入腦脊液，最終被腦啡肽酶和內(nèi)皮素轉(zhuǎn)換酶分解清除。在AD患者中，隨著pH值改變和載脂蛋白E的增多導(dǎo)致Aβ的產(chǎn)生和清除失衡，使Aβ在神經(jīng)元外聚集，發(fā)揮神經(jīng)毒性作用，最終引起記憶缺失[9-10]。研究表明，熱休克蛋白可以有效的促進(jìn)Aβ的清除，減少tau蛋白的過(guò)度磷酸化，減少神經(jīng)元的丟失，促進(jìn)神經(jīng)突觸的可塑性，發(fā)揮保護(hù)神經(jīng)細(xì)胞的作用[11-12]。 本研究中給P12神經(jīng)細(xì)胞及AD細(xì)胞分別加熱，均加熱60min/次，間隔24h重復(fù)加熱1次，其后將4組細(xì)胞與Aβ溶液進(jìn)行培養(yǎng)，熱療組細(xì)胞的存活率明顯高于非熱療組。研究表明熱療對(duì)神經(jīng)細(xì)胞均有明顯保護(hù)作用，熱療產(chǎn)生的HSP可以有效的減少Aβ在神經(jīng)元上的沉淀，促進(jìn)Aβ的清除，保護(hù)AD細(xì)胞。

深部熱療可以刺激神經(jīng)細(xì)胞產(chǎn)生大量熱休克蛋白，熱休克蛋白通過(guò)參與蛋白質(zhì)的合成、折疊、裝配、轉(zhuǎn)運(yùn)和降解等過(guò)程，以維持神經(jīng)細(xì)胞蛋白自穩(wěn)，提高細(xì)胞對(duì)應(yīng)激原的耐受性，恢復(fù)AD細(xì)胞正常的結(jié)構(gòu)和功能[13-14]。本研究試驗(yàn)在離體細(xì)胞水平探討AD的治療模式為每次AD細(xì)胞熱療的最佳時(shí)間為60min，間隔24h重復(fù)加熱1次，可以有效抑制Aβ聚集，從而保護(hù)神經(jīng)元細(xì)胞。未來(lái)，進(jìn)一步的研究中我們將繼續(xù)探索大鼠頭部熱療治療AD的模式和效果，并與離體細(xì)胞水平的治療相對(duì)比，我們期待可以在將來(lái)為深部熱療在AD患者的臨床治療提供前期的理論與實(shí)驗(yàn)基礎(chǔ)。

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The Exploration and Curative Effect of Deep Hyperthermia Treatment Model on Isolated AD Cells

CHEN Jianhong WANG Xiaopu NONG Haining BAO Jin LU Yimin*

Clifford Hospital, Guangzhou University of Chinese Medicine, Guangzhou 511495,China

Objective To explore the best treatment model of deep hyperthermia on isolated AD cells and evaluate its curative effect.Methods Isolated AD cells models were made ready and randomly divided into control group and hyperthermia control group, AD model group, AD model hyperthermia group. Each group was separately heated at the level of 41.5℃ for 30, 45, 60, 75, 90min，the absorbance(A) value was tested at 600 nm, and then the best hyperthermia treatment time was calculated. The ready made AD cells were heated for 60 minutes at the level of 41.5℃ and then continued to be incubated in thermotank for 24 hours at the level of 37℃ after the heating. The cells were separately taken out after 2, 4, 8, 12, 24 and 72h. Flow Cytometer was used to test the expression level of the AD cells HSP70, and the best interval of hyperthermia treatment was calculated. 4 groups of the cells were put into the Aβ25-3solution of 20μmol/L concentration to cultivate for 24 hours. The cell viability was tested by using flow cytometer to evaluate the hyperthermia treatment effects on AD. Results The control group and AD model group were separately given hyperthermia at the level of 41.5℃，the Cell proliferation trend of the two groups of cells began to decline compared with the non-heated cells(P<0.05). This trend came to the maximum at the 75th minute, and accordingly the safe and effective time length of heating on the cells was set for 60 minuets. The control hyperthermia compared with the AD model hyperthermia group, the Cell proliferation trend of the two groups which varied according to the temperature changes was generally the same, and no statistic significance was found between the two groups(P>0.05), which showed the best treatment time length of the two groups of cells was 60 minutes. The HSP70 expression level of AD model group was tested differently after the cells were heated 2, 4, 8, 12, 24 and 72h. The HSP70 expression level of AD cells was tested the top at 24h, then it began to decline. This showed the best treatment interval was 24 hours. The four groups of cells were mixed with Aβ and the mixture was then heated. MTT showed the viability of the four groups of cells was on the rising trend with temperature dependence(P<0.05), which showed hyperthermia could effectively reduce the gather of Aβ and help clear Aβ, AD cells were protected accordingly.Conclusions The best treatment time length of deep hyperthermia on isolated AD cells was 60 minutes, the best hyperthermia treatment interval was 24 hours, deep hyperthermia could effectively clear Aβ and help repair the neuron cells.

Hyperthermia; AD; Isolated Cell; Treatment Model

陳劍虹(1979 -)，男，漢族，本科，主治醫(yī)師，研究方向?yàn)槔夏瓴》较颉-mail：570686858@qq.com

盧宜民(1964 -)，男，漢族，本科，主任醫(yī)師，研究方向?yàn)槟[瘤熱療學(xué)。E-mail：gzlym-10@163.com

R-331

A

1007-8517(2017)15-0058-04

2017-05-25 編輯：梁志慶)