衛(wèi)哲

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電針對(duì)脊髓損傷后小鼠神經(jīng)功能重塑的機(jī)制研究

衛(wèi)哲1,2

(1.麗水學(xué)院醫(yī)學(xué)與健康學(xué)院,麗水 323000;2.汕頭大學(xué)醫(yī)學(xué)院神經(jīng)科學(xué)中心,汕頭 515041)

目的 觀察電針胸部夾脊穴對(duì)脊髓損傷后小鼠BMS評(píng)分、脊髓中巢蛋白表達(dá)和膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)表達(dá)的影響,探討電針的神經(jīng)保護(hù)和運(yùn)動(dòng)功能恢復(fù)機(jī)制。方法 將96只雌性C57BL/6小鼠隨機(jī)分為假手術(shù)組、脊髓損傷組、電針組、針刺組,每組24只。除假手術(shù)組外,其余3組給予T9水平脊髓損傷,電針組取T7和T11兩對(duì)夾脊穴進(jìn)行電針治療,針刺組取相同的穴位進(jìn)行單純手針治療。干預(yù)每次15 min,每日1次,每5 d休息1 d,共干預(yù)28 d。分別于干預(yù)后3 d、7 d、14 d、28 d評(píng)定各組小鼠的BMS評(píng)分變化,酶聯(lián)免疫法檢測(cè)脊髓中巢蛋白表達(dá),免疫熒光法、蛋白印跡法檢測(cè)脊髓中GFAP表達(dá)。結(jié)果 脊髓損傷后的14 d和28 d,電針組BMS主評(píng)分和副評(píng)分均明顯高于脊髓損傷組(＜0.05)。在損傷后28 d,針刺組BMS主評(píng)分和副評(píng)分均高于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05);電針組BMS副評(píng)分高于針刺組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05)。脊髓損傷后3 d、7 d和14 d電針組的巢蛋白含量明顯增多,與脊髓損傷組相比,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05)。脊髓損傷后7 d和14 d,針刺組的巢蛋白含量高于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05)。脊髓損傷后3 d和7 d,電針組的GFAP表達(dá)明顯高于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05);損傷后28 d,電針組和針刺組對(duì)GFAP免疫反應(yīng)性的抑制強(qiáng)于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05)。結(jié)論 電針夾脊穴可以促進(jìn)脊髓損傷后肢體功能恢復(fù),與電針7 d內(nèi)促進(jìn)巢蛋白和GFAP的表達(dá)有關(guān),而且二者表達(dá)呈正相關(guān)。電針治療的早期能促進(jìn)星形膠質(zhì)細(xì)胞的活化以發(fā)揮其神經(jīng)干細(xì)胞的作用,并在后期抑制GFAP的免疫反應(yīng)性,從而有利于神經(jīng)功能重塑。

電針;脊髓損傷;巢蛋白;BMS評(píng)分;膠質(zhì)纖維酸性蛋白;小鼠

脊髓損傷可以導(dǎo)致運(yùn)動(dòng)功能和感覺(jué)功能的缺失,會(huì)給患者帶來(lái)一系列精神上、社會(huì)上以及經(jīng)濟(jì)上的壓力[1]。深入闡釋脊髓損傷的分子機(jī)制,尋找出更有效的治療方法,從而提高患者的生活質(zhì)量,具有深遠(yuǎn)的臨床意義。顯而易見(jiàn),促進(jìn)脊髓損傷后運(yùn)動(dòng)功能的恢復(fù)是非常重要的。臨床常用皮質(zhì)甾類和神經(jīng)節(jié)苷脂等藥物治療脊髓損傷,急性脊髓損傷后常用皮質(zhì)類固醇激素作為臨床治療的首要干預(yù)方法。激素治療對(duì)于急性損傷后的抗炎作用明顯,但其對(duì)于促進(jìn)運(yùn)動(dòng)功能恢復(fù)的臨床意義還存在一定爭(zhēng)議。GM-1神經(jīng)節(jié)苷脂結(jié)合物理療法可以促進(jìn)不完全脊髓損傷后患者運(yùn)動(dòng)功能的恢復(fù)[2]。然而,迄今為止,由于藥物治療有明顯的副反應(yīng)、花費(fèi)高并且用藥周期長(zhǎng),還難以被廣大脊髓損傷患者所接受。近年來(lái),隨著細(xì)胞移植以及基因技術(shù)的發(fā)展,為脊髓損傷后神經(jīng)功能恢復(fù)帶來(lái)了新的希望,然而,其作為一種成熟技術(shù)應(yīng)用于臨床還為時(shí)過(guò)早[3-6]。

針灸作為傳統(tǒng)的中醫(yī)藥療法已經(jīng)普遍應(yīng)用于臨床多年,針灸可以促進(jìn)新陳代謝,提高機(jī)體免疫力,并且花費(fèi)低廉,易于操作,沒(méi)有任何副反應(yīng)。研究發(fā)現(xiàn),針灸有利于神經(jīng)損傷后的神經(jīng)系統(tǒng)機(jī)能恢復(fù)[7]。電針在臨床中應(yīng)用廣泛,針刺同時(shí)給予的低頻直流電治療,可以給機(jī)體傳導(dǎo)神經(jīng)沖動(dòng),在神經(jīng)突觸形成和可塑性中扮演著重要的角色。研究表明,電針可以改善脊髓損傷后運(yùn)動(dòng)功能及感覺(jué)功能的恢復(fù)[8]。然而,脊髓損傷后電針治療的分子機(jī)制尚需進(jìn)一步的研究,以便更好地為臨床治療提供理論依據(jù)。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物與分組

選取96只清潔級(jí)雌性C57BL/6小鼠,體重20～25 g,周齡10～12周,進(jìn)行適應(yīng)性喂養(yǎng)1個(gè)星期,飼養(yǎng)溫度與濕度分別為(23±2)℃和(60±10)%。動(dòng)物來(lái)源于黑龍江中醫(yī)藥大學(xué)動(dòng)物實(shí)驗(yàn)中心。將小鼠隨機(jī)分為假手術(shù)組、脊髓損傷組、脊髓損傷＋電針組(電針組)、脊髓損傷＋針刺組(針刺組)。

1.2 主要試劑和儀器

KWD808Ⅰ電脈沖治療儀(廣州英迪電子醫(yī)療設(shè)備公司),一次性使用無(wú)菌針灸針(0.25 mm×13 mm,中研太和醫(yī)藥公司), ELISA試劑盒(武漢博士德試劑公司),冰凍切片機(jī)(德國(guó)萊卡儀器公司),Axio顯微成像系統(tǒng)(德國(guó)蔡司公司),氯胺酮(福建寧德古田制藥公司),甲苯噻嗪(德國(guó)西格瑪公司),PBS緩沖液,4%多聚甲醛。

1.3 分組處理

假手術(shù)組僅僅給予小鼠椎板切除術(shù)。

脊髓損傷組給予小鼠T9水平的不完全脊髓損傷。

電針組給予小鼠T9水平的不完全脊髓損傷,第2日進(jìn)行電針治療,取穴方法參照《實(shí)驗(yàn)動(dòng)物學(xué)》及《針灸實(shí)驗(yàn)動(dòng)物圖譜》,選擇T7和T11兩對(duì)夾脊穴,針刺后連接KWD808Ⅰ電脈沖治療儀,疏密波(2/100 Hz),強(qiáng)度為0.2 mA,治療15 min,每日1次,每5 d休息1 d,共治療28 d。

針刺組給予小鼠T9水平的不完全脊髓損傷,第2日進(jìn)行單純手法針刺治療,取穴同電針組,每5 min捻針10 s,每日1次,每5 d休息1 d,共治療28 d。

1.4 灌注與取材

在治療的不同時(shí)間點(diǎn)(3 d,7 d,14 d,28 d),將小鼠用異戊烷吸入麻醉后,開(kāi)胸,輸液針刺入心尖部,灌注0.1 M PBS緩沖液(pH 7.4),剪開(kāi)左心耳,流出血液沖洗干凈后灌入4%多聚甲醛,直到臟器固定及四肢有僵硬感,或單獨(dú)灌注PBS緩沖液,取出脊髓,置于﹣80℃保存,根據(jù)實(shí)驗(yàn)安排,分別進(jìn)行酶聯(lián)免疫法、免疫熒光法、蛋白印跡法等測(cè)定。酶聯(lián)免疫的脊髓組織選擇以損傷處為中心上下端各0.5 cm的脊髓組織。免疫熒光的脊髓組織選擇損傷段以下0.5 cm脊髓組織。蛋白印跡的脊髓組織選擇以損傷為中心包括上下端0.5 cm的脊髓組織。

1.5 觀察指標(biāo)

1.5.1 BMS行為學(xué)評(píng)定

BMS主評(píng)分和副評(píng)分用來(lái)評(píng)價(jià)脊髓損傷后小鼠的運(yùn)動(dòng)功能[9-10]。測(cè)試由兩個(gè)有經(jīng)驗(yàn)的研究人員進(jìn)行操作。BMS主評(píng)分由0分(無(wú)任何踝部動(dòng)作)至9分(完全正常的運(yùn)動(dòng)能力)組成,BMS副評(píng)分總分為11分。分別在脊髓損傷后1 d、3 d、7 d、14 d、28 d進(jìn)行BMS行為學(xué)評(píng)定。

1.5.2 酶聯(lián)免疫法

將脊髓組織稱取重量后加入一定量的PBS,勻漿后離心20 min左右(3000 r/min),收集上清,分裝后待檢測(cè),具體操作步驟按照小鼠巢蛋白酶聯(lián)免疫分析試劑盒進(jìn)行。

1.5.3 免疫熒光法

在脊髓損傷后3 d、28 d以4%多聚甲醛灌注取材后,分離脊髓組織以4%多聚甲醛浸泡過(guò)夜,次日置于含有30%蔗糖的PB溶液中于室溫?cái)R置1 d進(jìn)行高滲脫水。次日取出脊髓組織以O(shè)CT包埋,冰凍切片機(jī)上連續(xù)縱向切片(厚度為15mm),0.01 M枸緣酸鈉溶液(pH 6.0)99℃水浴40 min,PBS洗1次,10%驢血清含0.2%的Triton X-100室溫封閉1 h,10%驢血清加入兔抗多克隆膠質(zhì)酸性纖維蛋白抗體(1:2000)作為一抗,均勻覆蓋于脊髓組織切片上,置于濕盒中于4℃冰箱過(guò)夜孵育。次日,棄去未結(jié)合的一抗,PBS清洗3次,每次5 min,加入抗一抗種屬來(lái)源的紅色熒光二抗(激發(fā)光波長(zhǎng)為594 nm),置于濕盒室溫孵育1 h。棄去未結(jié)合的二抗溶液,PBS清洗3次,每次5 min,加入含有DAPI的PBS溶液,細(xì)胞核染色5 min,使用水溶性封片液,蓋玻片封片。

1.5.4 蛋白印跡

RIPA蛋白抽提試劑,加入蛋白酶抑制劑及0.1 M PMSF(比例1:9)。稱取組織以稱重,加入裂解液,電動(dòng)組織勻漿器15000 r/min轉(zhuǎn)速進(jìn)行勻漿,4℃離心,13000 r/min,20 min。離心完成后取上清,分裝保存,待測(cè)。以BCA法測(cè)蛋白濃度,以RIPA調(diào)整蛋白濃度,蛋白煮沸變性10 min。配備12%的分離膠,濃縮膠濃度為5%,待測(cè)蛋白樣品上樣量為10mg/孔,電泳條件選擇80～120 V。選擇濕轉(zhuǎn)法轉(zhuǎn)膜,轉(zhuǎn)膜條件為300 mA,時(shí)間為3 h,轉(zhuǎn)膜后麗春紅染色試劑對(duì)膜進(jìn)行染色,觀察轉(zhuǎn)膜效果。將膜置于3%BSA-TBST中室溫輕搖1 h封閉。將膜以3%BSA-TBST稀釋膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)及GAPDH (1:1000)一抗孵育,于4℃冰箱中輕搖過(guò)夜。次日, TBST洗膜3次,每次5 min,3%BSA-TBST稀釋抗一抗種屬來(lái)源的二抗(1:1000),室溫輕搖1 h,TBST洗膜3次,每次5 min。ECL顯色液顯色,曝光,使用儀器為alpha凝膠成像系統(tǒng)。

1.6 統(tǒng)計(jì)學(xué)方法

所有數(shù)據(jù)由SPSS19.0統(tǒng)計(jì)軟件包處理。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差表示,采用單因素方差分析、檢驗(yàn)。以＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 電針對(duì)脊髓損傷后小鼠BMS評(píng)分的影響

由圖1可見(jiàn),脊髓損傷后的第1天,除假手術(shù)組外,其余3組小鼠的BMS評(píng)分明顯降低,并在脊髓損傷3 d后開(kāi)始逐漸增高。在損傷后14 d和28 d,電針組BMS主評(píng)分和副評(píng)分均明顯高于脊髓損傷組(＜0.05)。在損傷后28 d,針刺組BMS主評(píng)分和副評(píng)分均高于脊髓損傷組,差異有統(tǒng)計(jì)學(xué)意義(＜0.05);電針組BMS副評(píng)分高于針刺組,差異有統(tǒng)計(jì)學(xué)意義(＜0.05)。而在其他時(shí)間點(diǎn)組別之間無(wú)明顯差異。說(shuō)明電針與針刺均可提高脊髓損傷后小鼠BMS評(píng)分,并且電針組優(yōu)于針刺組。

2.2 電針對(duì)脊髓損傷后GFAP表達(dá)的影響

免疫熒光法測(cè)定GFAP表達(dá)結(jié)果顯示,脊髓損傷后3 d,與脊髓損傷組比較,電針組損傷部位出現(xiàn)明顯的神經(jīng)膠質(zhì)增多(圖2A),脊髓損傷組GFAP表達(dá)增多,電針組GFAP表達(dá)高于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05);脊髓損傷后28 d,電針組和針刺組GFAP表達(dá)明顯低于脊髓損傷組,差異有統(tǒng)計(jì)學(xué)意義(＜0.05) (圖2B)。蛋白印跡法測(cè)定GFAP表達(dá)結(jié)果顯示,脊髓損傷后3 d和7 d,電針組GFAP表達(dá)明顯高于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05);損傷后28 d,電針組和針刺組對(duì)GFAP免疫反應(yīng)性的抑制強(qiáng)于脊髓損傷組,差異具有統(tǒng)計(jì)學(xué)意義(＜0.05)(圖2C、D)。

注:電針組與脊髓損傷組比較1)P＜0.05;針刺組與脊髓損傷組比較2)P＜0.05;電針組與針刺組比較3)P＜0.05

注:電針組與脊髓損傷組比較1)P＜0.05;針刺組與脊髓損傷組比較2)P＜0.05

2.3 電針對(duì)脊髓損傷后脊髓中巢蛋白表達(dá)的影響

脊髓損傷組小鼠巢蛋白含量在脊髓損傷后7 d開(kāi)始增高并在損傷后14 d出現(xiàn)降低。與脊髓損傷組相比,脊髓損傷后3 d、7 d和14 d電針組的巢蛋白含量明顯增多,差異有統(tǒng)計(jì)學(xué)意義(＜0.05)。脊髓損傷后7 d和14 d,針刺組的巢蛋白含量高于脊髓損傷組,差異有統(tǒng)計(jì)學(xué)意義(＜0.05)。然而,脊髓損傷后28 d,除了假手術(shù)組,各組巢蛋白含量減少,并且3組之間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(＞0.05)。詳見(jiàn)圖3。

注:電針組與脊髓損傷組比較1)P＜0.05;針刺組與脊髓損傷組比較2)P＜0.05

3 討論

根據(jù)中醫(yī)學(xué)的臟腑經(jīng)絡(luò)理論,脊髓損傷的病理學(xué)基礎(chǔ)為督脈損傷。傳統(tǒng)中醫(yī)學(xué)認(rèn)為,華佗夾脊穴位于脊柱兩旁,內(nèi)夾督脈,外貫足太陽(yáng)膀胱經(jīng),一方面可以通暢脊背經(jīng)氣,一方面可以通過(guò)督脈及多血少氣之膀胱經(jīng)發(fā)揮調(diào)節(jié)全身氣血的作用[11-12]?，F(xiàn)代中醫(yī)學(xué)以?shī)A脊穴的解剖學(xué)位置為基礎(chǔ),以脊椎與神經(jīng)的節(jié)段性分布關(guān)系為依據(jù),選取相應(yīng)的夾脊穴治療該節(jié)段神經(jīng)支配的相關(guān)病癥,并取得良好療效。研究表明,電針夾脊穴治療對(duì)中樞神經(jīng)系統(tǒng)損傷、椎間盤(pán)病變、相應(yīng)臟器病變起到良好療效。研究發(fā)現(xiàn),電針夾脊穴可以提高脊髓損傷后大鼠下肢的BBB評(píng)分,縮短皮層體感誘發(fā)電位潛伏期,促進(jìn)神經(jīng)損傷的功能恢復(fù)[13]。電針夾脊穴對(duì)脊髓損傷后表皮生長(zhǎng)因子、NG2蛋白等多種分子的表達(dá)起到促進(jìn)作用[14-15]。然而,電針夾脊穴對(duì)脊髓損傷后的分子機(jī)制研究尚需進(jìn)一步明確?；诖?我們建立脊髓損傷小鼠模型,選擇以脊髓損傷段為中心的上下兩組夾脊穴進(jìn)行電針治療。

電針能夠?qū)C(jī)體產(chǎn)生神經(jīng)沖動(dòng),在突觸形成和穩(wěn)定性中發(fā)揮著重要作用。前期研究表明,電針可以調(diào)節(jié)脊髓損傷后神經(jīng)生長(zhǎng)因子和炎性因子的表達(dá)從而促進(jìn)神經(jīng)再生[16-18]。本研究中,課題組再次證實(shí)電針和針刺均可促進(jìn)脊髓損傷后下肢運(yùn)動(dòng)功能的恢復(fù),并且電針組療效優(yōu)于針刺組。課題組選擇疏密波(2/100 Hz)作為電針波形,能夠克服由單一波形帶來(lái)的機(jī)體易適應(yīng)性。

巢蛋白是神經(jīng)干細(xì)胞的標(biāo)記蛋白,和細(xì)胞分化密切相關(guān)[19]。本研究發(fā)現(xiàn),在脊髓損傷后的早期(脊髓損傷后3 d、7 d),電針可以促進(jìn)大量的巢蛋白表達(dá)。損傷后14 d,電針組和針刺組的巢蛋白含量出現(xiàn)降低,但仍然高于脊髓損傷組(＜0.05)。結(jié)果表明,電針對(duì)脊髓損傷后早期發(fā)揮著促進(jìn)神經(jīng)元分化和增殖的作用。因此,電針激活成年哺乳動(dòng)物的神經(jīng)干細(xì)胞功能極有可能在神經(jīng)細(xì)胞再生和組織修復(fù)的過(guò)程中發(fā)揮重要作用。GFAP僅存在于星形膠質(zhì)細(xì)胞,并促進(jìn)神經(jīng)元生長(zhǎng)[20]。在成年哺乳動(dòng)物中,GFAP對(duì)神經(jīng)系統(tǒng)起到支持、營(yíng)養(yǎng)、隔離和保護(hù)的作用。星形膠質(zhì)細(xì)胞的過(guò)度激活會(huì)引起疾病的發(fā)生。逐漸地,星形膠質(zhì)細(xì)胞的過(guò)度肥大、增殖和遷移促進(jìn)神經(jīng)膠質(zhì)瘢痕形成[21-23]。神經(jīng)膠質(zhì)瘢痕形成的越多,對(duì)損傷后神經(jīng)恢復(fù)的阻礙就越大。本次研究結(jié)果顯示,GFAP的免疫反應(yīng)性表明在脊髓損傷后早期(脊髓損傷后3 d、7 d),電針促進(jìn)星形膠質(zhì)細(xì)胞活化,而在損傷的后期(脊髓損傷后28 d),電針促進(jìn)星形膠質(zhì)細(xì)胞活化水平降低。

GFAP和巢蛋白的表達(dá)正相關(guān),電針對(duì)二者作用相似,共同促進(jìn)損傷后神經(jīng)功能的重建。本研究發(fā)現(xiàn),電針可以促進(jìn)星形膠質(zhì)細(xì)胞的形成從而在神經(jīng)干細(xì)胞中發(fā)揮作用。然而,電針對(duì)神經(jīng)損傷后神經(jīng)保護(hù)的具體分子機(jī)制還有待于進(jìn)一步闡述。

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Mechanism Study on Electroacupuncture in Reconstructing the Neurologic Function in Mice after Spinal Cord Injury

1,2.

1.,,323000,; 2.,,515041,

Objective To observe the effect of electroacupuncture at thoracic Jiaji points (EX-B 2) on the Behavior Measurement Scale (BMS) score, and expressions of nestin and glial fibrillary acidic protein (GFAP) in spinal cord in mice after spinal cord injury, and to explore the action mechanism of electroacupuncture in protecting nerves and recovering motor function. Method Ninety-six female C57BL/6 mice were randomized into a sham operation group, a spinal cord injury group, an electroacupuncture group, and an acupuncture group, 24 mice in each group. Except for the sham operation group, spinal cord injury at T9level was induced in the other three groups. The electroacupuncture group was intervened by electroacupuncture at Jiaji points (EX-B 2) of T7and T11, and the acupuncture group received manual acupuncture at the same acupoints. The intervention was conducted 15 min each time, once a day, with 1 d interval every 5 d, for a total of 28 d. The BMS score was evaluated respectively after 3 d, 7 d, 14 d, and 28 d intervention. Enzyme-linked immunosorbent assay (ELISA) was adopted to detect the expression of nestin in spinal cord, and immunofluorescence and Western blotting were used to detect the expression of GFAP in spinal cord. Result Respectively 14 d and 28 d after spinal cord injury, the main and auxiliary scores of BMS in the acupuncture group were significantly higher than those in the spinal cord injury group (＜0.05); 28 d after spinal cord injury, the main and auxiliary scores of BMS in the acupuncture group were significantly higher than those in the spinal cord injury group (＜0.05) and the auxiliary BMS score in the electroacupuncture group was significantly higher than that in the acupuncture group (＜0.05). Respectively 3 d, 7 d, and 14 d after spinal cord injury, the contents of nestin in the electroacupuncture group were obviously increased and significantly different from those in the spinal cord injury group (＜0.05). The nestin content of the acupuncture group was significantly higher than that of the spinal cord injury group 7 d and 14 d after spinal cord injury (＜0.05). The expression of GFAP in the electro- acupuncture group was significantly higher than that in the spinal cord injury group 3 d and 7 d after spinal cord injury (＜0.05); the inhibitions on the immunoreactivity of GFAP in the electroacupuncture group and acupuncture group were more significant than the inhibition in the spinal cord injury group 28 d after the injury (＜0.05). Conclusion Electroacupuncture at Jiaji points (EX-B 2) can promote the recovery of motor function after spinal cord injury, which is related to the enhancement of the expressions of nestin and GFAP within 7 d after the injury, and the two expressions are in a positive correlation. In the early stage of treatment, electroacupuncture can boost the activity of astrocytes to act as neural stem cells and inhibit the immunoreactivity of GFAP in the later stage to benefit the reconstruction of neurologic function.

Electroacupuncture; Spinal cord injury; Nestin; BMS; Glial fibrillary acidic protein; Mice

1005-0957(2017)05-0608-06

R2-03

A

10.13460/j.issn.1005-0957.2017.05.0608

國(guó)家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(81503639);黑龍江省自然科學(xué)基金面上項(xiàng)目(H2016070)

衛(wèi)哲(1981—),女,副主任醫(yī)師,博士后,Email:weizhe315@126.com

2016-11-24