金俊健

電針刺激后大鼠血清對(duì)成骨細(xì)胞OPG、RANKL mRNA及其蛋白表達(dá)的影響

金俊健

(臨海市第二人民醫(yī)院,臨海 317016)

觀察電針刺激去卵巢骨質(zhì)疏松癥模型大鼠的血清對(duì)體外培養(yǎng)成骨細(xì)胞的骨形成因子骨保護(hù)素(osteoprotegerin, OPG)和骨吸收因子(receptor activator for nuclear factor-kB ligand, RANKL)mRNA表達(dá)及其蛋白濃度的影響。將45只雌性SD大鼠,隨機(jī)分為空白組(A組)、模型組(B組)、電針刺激組(C組),每組15只。除A組外,B組、C組均切除雙側(cè)卵巢。造模成功后,C組給予電針刺激,B組正常飼養(yǎng),A組不做任何處理,常規(guī)正常飼養(yǎng)。12周后,采用水合氯醛麻醉各組大鼠,心臟采血,經(jīng)過(guò)處理后加到體外培養(yǎng)的大鼠成骨細(xì)胞培養(yǎng)液中。采用堿性磷酸酶(ALP)檢測(cè)成骨細(xì)胞活性,RT-qPCR法檢測(cè)大鼠血清對(duì)OPG、RANKL mRNA表達(dá)的影響,ELISA法檢測(cè)大鼠血清對(duì)OPG、RANKL蛋白濃度的影響。各組大鼠血清處理成骨細(xì)胞后,與A組比較,B組ALP的活性明顯降低(＜0.05),C組電刺激后成骨細(xì)胞內(nèi)ALP的活性明顯增加(＜0.05)。與B組比較,A組及C組OPG mRNA表達(dá)及其蛋白濃度表達(dá)明顯降低(＜0.05);與A組比較,C組OPG mRNA表達(dá)及其蛋白濃度均明顯降低(＜0.05)。與B組比較,C組及A組RANKL mRNA表達(dá)及其蛋白濃度表達(dá)明顯降低(＜0.05);與A組比較,C組RANKL mRNA及蛋白濃度明顯降低(＜0.05)。C組大鼠骨密度明顯高于B組(＜0.05)。電刺激后的大鼠血清可以提升ALP、OPG水平,降低RANKL水平,其治療骨質(zhì)疏松癥的機(jī)制可能與此有關(guān)。

針刺療法;電針;骨質(zhì)疏松;成骨細(xì)胞;骨保護(hù)素;骨吸收因子;大鼠

隨著我國(guó)人口老齡化現(xiàn)象的加劇,患骨質(zhì)疏松癥的人數(shù)在逐年增加[1],其嚴(yán)重威脅人們的身體健康及生存生活質(zhì)量[2],因此積極尋找有效的防治方法具有重要意義。中醫(yī)是我國(guó)的瑰寶,其中應(yīng)用針灸療法治療一些疾病具有顯著療效,近年來(lái)發(fā)現(xiàn)針灸治療骨質(zhì)疏松癥具有一定的治療效果[3],并且通過(guò)電針刺激法治療骨質(zhì)疏松癥也已經(jīng)成為國(guó)內(nèi)外專(zhuān)家學(xué)者的研究點(diǎn)。在骨質(zhì)疏松癥發(fā)生發(fā)展過(guò)程中,OPG、RANKL系統(tǒng)在成骨細(xì)胞與破骨細(xì)胞相互調(diào)節(jié)的過(guò)程中起著重要作用。本文旨在通過(guò)研究電針刺激后的大鼠血清對(duì)體外培養(yǎng)的大鼠成骨細(xì)胞OPG、RANKL蛋白及mRNA表達(dá)的影響,探討電針刺激療法治療骨質(zhì)疏松癥的機(jī)制。

1 材料和方法

1.1 實(shí)驗(yàn)動(dòng)物與分組

采用3月齡SD大鼠45只(90 d),雌性,體質(zhì)量為(280±20)g;SD乳鼠10只,購(gòu)自北京維通利華實(shí)驗(yàn)技術(shù)有限公司[許可證號(hào)SCXK(京)2011-0011],飼養(yǎng)溫度為25℃～27℃,濕度保持在50%～70%。實(shí)驗(yàn)對(duì)動(dòng)物的處理方法符合中華人民共和國(guó)科學(xué)技術(shù)部頒發(fā)的《關(guān)于善待實(shí)驗(yàn)動(dòng)物的指導(dǎo)性意見(jiàn)》。實(shí)驗(yàn)地點(diǎn)為臨海市第二人民醫(yī)院動(dòng)物實(shí)驗(yàn)中心。動(dòng)物適應(yīng)性飼養(yǎng)1周后,將45只大鼠隨機(jī)分為空白組(A組)15只和造模組30只。造模成功后,造模組再隨機(jī)分為2組,模型組(B組)15只,電針刺激組(C組)15只。

1.2 主要試劑與儀器

10%水合氯醛(山東嘉穎化工科技有限公司);胎牛血清、DMEM培養(yǎng)基、胰酶、雙抗(Gibico,美國(guó));大鼠OPG、RANKL ELISA試劑盒(中國(guó)武漢博士德生物工程有限公司);堿性磷酸酶(ALP)試劑盒(南京建成生物工程研究所);RT-PCR試劑盒(日本,Takara)。

電針儀(廣州市健玲醫(yī)療器械有限公司);多功能酶標(biāo)儀(美國(guó)Bio-Rad公司);恒溫箱(北京長(zhǎng)安儀器廠);二氧化碳培養(yǎng)箱(Thermo Scientific);低溫離心機(jī)型(美國(guó)Sigma公司);大鼠成骨細(xì)胞專(zhuān)用培養(yǎng)基(武漢普諾賽生命科技有限公司);PCR儀(美國(guó)ABI公司);微量移液器等。

1.3 模型建立[4]

大鼠腹腔注射10%水合氯醛(3 mL/kg)進(jìn)行麻醉,固定于手術(shù)臺(tái)上,經(jīng)背部正中切口入路切除雙側(cè)卵巢后,逐層關(guān)閉,縫合肌肉、皮膚。乙醇消毒后,術(shù)后肌肉注射青霉素預(yù)防感染。術(shù)后第2天連續(xù)3 d做陰道細(xì)胞涂片,經(jīng)證實(shí)卵巢切除完全,采用QDR4500A型雙能X線骨密度測(cè)定儀測(cè)定大鼠骨密度(BMD,美國(guó)歐力士),采用放射免疫法測(cè)定血清雌二醇(BE,試劑盒購(gòu)自上海信帆生物科技有限公司),大鼠BMD及BE明顯降低,復(fù)制骨質(zhì)疏松癥模型成功。

A組不做任何處理,常規(guī)飼養(yǎng)。

B組造模成功后,常規(guī)飼養(yǎng)。

C組模型成功后,參照丁巖等[5]電針刺激方法干預(yù)大鼠,選背部L1-4夾脊、環(huán)跳(右側(cè))、足三里穴進(jìn)行針刺。針刺深度為6～8 mm,針身接刺激儀指數(shù)曲線波3 Hz,強(qiáng)度控制在局部可見(jiàn)輕微肌肉收縮,留針刺激20 min。隔日1次,持續(xù)12周。

1.4 大鼠成骨細(xì)胞的體外培養(yǎng)

將SD乳鼠置于75%乙醇中浸泡,在無(wú)菌條件下取下顱骨(除去骨膜、結(jié)締組織),用PBS清洗3次后將顱骨剪碎成約1 mm3。將骨片轉(zhuǎn)到裝有0.25%胰酶培養(yǎng)瓶中,消化完畢后去掉上清液;再用0.1%的Ⅱ型膠原酶消化收集合并上清液,終止消化,并將其用200目濾網(wǎng)過(guò)濾3次,1000 r/min離心10 min,棄掉上清,加入培養(yǎng)基制成細(xì)胞混懸液,接種于培養(yǎng)瓶,于5% CO2培養(yǎng)箱中37℃條件下培養(yǎng),每3 d換液1次,直到80%細(xì)胞融合。細(xì)胞進(jìn)行常規(guī)消化、傳代,收集第二代細(xì)胞用于實(shí)驗(yàn)。

1.5 成骨細(xì)胞的血清處理方法

10%水合氯醛(3 mL/kg)麻醉各組大鼠后,心臟取血,冷置1 h,2500 r/min離心25 min,棄去沉淀物,保留上層血清。將上層血清采用56℃水浴滅活30 min,并用0.22mm的濾網(wǎng)抽濾除菌。將各組制備的血清按照10%的比例加入成骨細(xì)胞中培養(yǎng)3 d。

1.6 觀察指標(biāo)

1.6.1 ALP活性檢測(cè)

收集各組血清處理后的成骨細(xì)胞上清液,按照試劑盒說(shuō)明書(shū)操作,檢測(cè)ALP活性。

1.6.2 RT-qPCR法檢測(cè)成骨細(xì)胞OPG、RANKLmRNA表達(dá)的影響

收集各組血清處理后的成骨細(xì)胞上清液,提取細(xì)胞總RNA,于PCR儀中行逆轉(zhuǎn)錄,按照預(yù)先設(shè)定熒光定量PCR反應(yīng)程序進(jìn)行擴(kuò)增,計(jì)算OPG、RANKL基因與GAPDH基因擴(kuò)增條帶表達(dá)量的比值表示。

1.6.3 ELISA法檢測(cè)成骨細(xì)胞OPG、RANKL蛋白濃度的影響

收集各組血清處理后的成骨細(xì)胞上清液,嚴(yán)格按照試劑盒說(shuō)明書(shū)操作。

1.7 統(tǒng)計(jì)學(xué)方法

所有數(shù)據(jù)采用SPSS19.0軟件進(jìn)行統(tǒng)計(jì)處理。符合正態(tài)分布的計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,多組間比較采用單因素方差分析,組間比較采用檢驗(yàn)。以＜0.05表示差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組大鼠骨密度測(cè)定

A組大鼠的骨密度值為(0.22±0.06)g/cm2,B組大鼠的骨密度值為(0.12±0.05)g/cm2,C組大鼠的骨密度值為(0.16±0.06))g/cm2,3組大鼠骨密度值比較差異有統(tǒng)計(jì)學(xué)意義(=11.603,=0.000),其中A組骨密度明顯高于B組及C組(=6.811、3.406,均＜0.05),C組明顯高于B組(=3.406,＜0.05)。

2.2 各組大鼠血清對(duì)成骨細(xì)胞ALP活性的影響

表1 各組大鼠成骨細(xì)胞ALP活性比較 (±s,U/gprot)

注:與B組比較1)＜0.05

3組成骨細(xì)胞ALP活性比較,差異具有統(tǒng)計(jì)學(xué)意義(=476.75,＜0.01)。其中A組、C組大鼠成骨細(xì)胞ALP的活性均明顯高于B組(=39.127,＜0.05;=36.359,＜0.05)。詳見(jiàn)表1、圖1。

注:與A組比較*P＜0.05;與B組比較#P＜0.05

2.3 各組大鼠血清OPG、RANKL mRNA表達(dá)比較

與B組比較,A組及C組OPG mRNA表達(dá)明顯降低(=55.578、34.064,＜0.05),與A組比較,C組OPG mRNA表達(dá)明顯降低(=21.514,＜0.05)。與B組比較,A組及C組RANKL mRNA表達(dá)明顯升高(=47.881、52.234,＜0.05),與A組比較,C組RANKL mRNA表達(dá)顯著升高(=4.353,＜0.05)。與B組比較,A組及C組OPG/RANKL比值明顯升高(=18.006、7.982,＜0.05),與A組比較, C組OPG/RANKL比值明顯降低(=10.024,＜0.05)。詳見(jiàn)表2。

表2 各組大鼠成骨細(xì)胞OPG、RANKL mRNA表達(dá)比較 (±s)

注:與A組比較1)＜0.05;與B組比較2)＜0.05

2.4 各組大鼠血清OPG、RANKL蛋白濃度比較

表3 各組大鼠成骨細(xì)胞OPG、RANKL蛋白濃度表達(dá)比較 (±s)

注:與A組比較1)＜0.05;與B組比較2)＜0.05

相較于B組,A組及C組RANKL蛋白濃度均明顯升高(=70.048、57.418,均＜0.05);與A組比較,C組RANKL蛋白濃度顯著降低(=12.630,＜0.05)。與B組比較,A組及C組OPG蛋白濃度明顯降低(=30.168,＜0.05;= 21.589,＜0.05);與A組比較,C組OPG蛋白濃度顯著降低(=8.580,＜0.05)。與B組比較,A組及C組OPG/ RANKL比值均明顯升高(=24.186，＜0.05;=9.315,＜0.05);與A組比較,C組OPG/RANKL比值明顯降低(=14.871,＜0.05)。詳見(jiàn)表3。

3 討論

有研究報(bào)道,通過(guò)探究生化、骨密度、生物力學(xué)、蛋白和基因等一些指標(biāo),表明針灸、電針刺激可以治療骨質(zhì)疏松癥[6-9]。電刺激對(duì)成骨細(xì)胞有增殖的作用,可以影響骨的重建,而治療骨質(zhì)疏松癥[10]。電刺激治療骨質(zhì)疏松癥的機(jī)制可能是,調(diào)節(jié)破骨細(xì)胞骨破壞,促進(jìn)成骨細(xì)胞的增殖與分化,影響骨的代謝,從而起到促進(jìn)骨形成、抑制骨吸收,達(dá)到預(yù)防和治療骨質(zhì)疏松癥的效果[11]。成骨細(xì)胞在骨組織的生長(zhǎng)、發(fā)展過(guò)程中發(fā)揮著重要作用,可以合成、分泌骨基質(zhì),因此促進(jìn)成骨細(xì)胞的成骨功能是防治骨質(zhì)疏松癥的主要療法之一。本實(shí)驗(yàn)通過(guò)電針刺激血清學(xué)的方法,在蛋白及基因水平上進(jìn)一步探究其對(duì)成骨細(xì)胞的骨形成及骨吸收的影響。

ALP是成骨細(xì)胞分化的一種酶蛋白,是成骨細(xì)胞分化的早期指標(biāo),成骨細(xì)胞分化越多ALP的表達(dá)就越多,ALP活性越高成骨細(xì)胞的形成越好,是骨形成指標(biāo)。本實(shí)驗(yàn)結(jié)果顯示,B組大鼠血清作用于體外培養(yǎng)的成骨細(xì)胞時(shí)相較于A組,ALP的活性明顯下降,電針刺激后可以增加ALP的活性,提示電針刺激療法能促進(jìn)成骨細(xì)胞的分化,促進(jìn)骨形成。

OPG、RANKL屬于腫瘤壞死因子(TNF)家族,由成骨細(xì)胞表達(dá),是破骨細(xì)胞分化、調(diào)節(jié)的信號(hào)因子,通過(guò)調(diào)控OPG、RANKL的表達(dá)可以調(diào)控骨吸收,是骨吸收、骨重建的重要因素[12-15]。成骨細(xì)胞可以通過(guò)分泌OPG、RANKL, OPG可與RANKL競(jìng)爭(zhēng)RANK受體抑制破骨的形成及活性[16-19]。OPG是一種可溶性分泌型糖蛋白在成骨細(xì)胞中高表達(dá)[20],是RANKL的受體,可以與破骨細(xì)胞表面的RANK競(jìng)爭(zhēng)性結(jié)合RANKL,阻斷骨吸收信號(hào)傳遞以及破骨細(xì)胞的分化,抑制破骨細(xì)胞吸收活性。本研究結(jié)果顯示,電針刺激后大鼠血清可以上調(diào)成骨細(xì)胞OPG mRNA的表達(dá)及蛋白濃度,提示電針刺激療法可能通過(guò)促進(jìn)成骨細(xì)胞OPG的表達(dá),從而增加OPG與RANKL的結(jié)合,抑制破骨細(xì)胞的活性,以此來(lái)達(dá)到治療骨質(zhì)疏松癥的效果。RANKL是影響骨吸收的關(guān)鍵因子,與破骨細(xì)胞表面的RANK結(jié)合,促進(jìn)破骨細(xì)胞的分化,抑制其凋亡[21]。本實(shí)驗(yàn)結(jié)果顯示,與A組血清相比,B組血清成骨細(xì)胞RANKL mRNA的表達(dá)及蛋白濃度明顯升高,電針刺激后RANKL蛋白、mRNA顯著下調(diào),提示電針刺激療法后的大鼠血清可能通過(guò)抑制RANKL的表達(dá),來(lái)抑制破骨細(xì)胞的功能,從而治療骨質(zhì)疏松癥。OPG/RANKL在機(jī)體中處于動(dòng)態(tài)平衡,當(dāng)比值下降時(shí),會(huì)引起一些骨代謝疾病,而當(dāng)比值升高時(shí),會(huì)減弱破骨細(xì)胞的分化成熟[22]。在本實(shí)驗(yàn)中,電針刺激后大鼠血清作用于體外培養(yǎng)的成骨細(xì)胞可以使OPG/RANKL的比值增加,抑制破骨細(xì)胞分化,增加骨形成。對(duì)大鼠骨密度測(cè)試顯示,電針刺激對(duì)大鼠的骨密度下降程度有所抑制。

目前,治療骨質(zhì)疏松癥的方法主要是以藥物為主,患者又大部分是老年人,由于年齡及身體因素的影響,對(duì)藥物吸收較差,受副作用影響較大。OPG/RANKL/RANK信號(hào)系統(tǒng)是骨代謝的重要信號(hào)通路,是骨代謝疾病治療的新靶點(diǎn)。

綜上所述,骨質(zhì)疏松癥模型大鼠血清使成骨細(xì)胞ALP、OPG水平明顯下降,RANKL水平增高,且OPG/RANKL顯著下降,電刺激后的大鼠血清可以提升ALP、OPG水平,降低RANKL水平,調(diào)節(jié)OPG/RANKL的比值,在成骨細(xì)胞重建上,電針刺激后大鼠血清能影響OPG與RANKL的蛋白、mRNA水平表達(dá),是治療骨質(zhì)疏松癥的一種治療方法,因此電針刺激療法的應(yīng)用將具有重要意義。

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Effect of Rat Serum after Electroacupuncture Stimulation on the Expressions of Osteoblastic OPG and RANKL mRNAs and Their Proteins

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317016,

To investigate the effect of ovariectomized osteoporosis rat serum after electroacupuncture stimulation on the expressions of in vitro cultured osteoblastic bone formation factor osteoprotegerin (OPG) and bone resorption factor receptor activator of nuclear factor-kB ligand (RANKL) mRNAs and the concentrations of their proteins.Forty-five female SD rats were randomized to a blank group (group A), a model group (group B) and an electroacupuncture stimulation group (group C), 15 rats each. Bilateral ovaries were excised in groups B and C except group A. After successful model making, group C was given electroacupuncture stimulation and group B, fed regularly. Group A was fed regularly without any treatment. After 12 weeks, each group of rats was anesthetized with chloral hydrate and blood was taken from the heart. The blood was processed and put in culture fluid for in vitro cultured rat osteoblasts. Osteoblast activity was measured using alkaline phosphatase (ALP). The effect of rat serum on the expressions of osteoblastic OPG and RANKL mRNAs was assessed using RT-qPCR. The effect of rat serum on the concentrations of OPG and RANKL proteins was determined using ELISA.After osteoblasts were treated with rat serumin in each group of rats, ALP activity decreased significantly in group B (＜0.05), and osteoblastic ALP activity increased significantly in group C after electroacupuncture stimulation (＜0.05), in comparison with group A. OPG mRNA expression and its protein concentration decreased significantly in groups A and C compared with group B (＜0.05) and in group C compared with group A (＜0.05). RANKL mRNA expression and its protein concentration decreased significantly in groups C and A compared with group B (＜0.05) and in group C compared with group A (＜0.05). Bone mineral density was significantly higher in group C of rats than in group B (＜0.05).Rat serum after electroacupuncture stimulation can increase ALP and OPG levels and decrease RANKL levels, which may be related to the mechanism by which electroacupuncture treats osteoporosis.

Acupuncture therapy; Electroacupuncture; Osteoporosis; Osteoblast; Osteoprotegerin; Bone resorption factor; Rats

R2-03

A

10.13460/j.issn.1005-0957.2019.07.0798

1005-0957(2019)07-0798-05

2019-01-10

金俊健(1976—),男,主治醫(yī)師