褚 浩,王勤章,吳 雙,錢(qián) 成,王議鶴,錢(qián) 彪
·論著·
納米細(xì)菌大鼠腎結(jié)石模型腎臟結(jié)石形成時(shí)間的動(dòng)態(tài)研究
褚 浩,王勤章,吳 雙,錢(qián) 成,王議鶴,錢(qián) 彪*
目的 采用微計(jì)算機(jī)斷層掃描技術(shù)(Mirco-CT)和病理組織學(xué)檢查動(dòng)態(tài)觀察納米細(xì)菌誘導(dǎo)的大鼠腎結(jié)石模型腎臟結(jié)石形成情況。方法 2015年11月—2016年6月,將60只SPF級(jí)Wistar大鼠適應(yīng)性飼養(yǎng)1周后,隨機(jī)分為對(duì)照組和誘石組,各30只。對(duì)照組大鼠一次性尾靜脈注射0.9%氯化鈉溶液1.2 ml,誘石組大鼠一次性尾靜脈注射納米細(xì)菌懸液1.2 ml。兩組大鼠注射后第1~10周每周處死3只,收集雙側(cè)腎臟待測(cè)。采用Mirco-CT檢查兩組大鼠腎臟,統(tǒng)計(jì)第1~10周兩組大鼠檢測(cè)到的腎臟高密度影情況。光學(xué)顯微鏡下觀察兩組大鼠腎臟晶體的形成情況,統(tǒng)計(jì)第1~10周兩組大鼠腎臟晶體陽(yáng)性情況。結(jié)果 Mirco-CT掃描結(jié)果:第1~10周,對(duì)照組大鼠腎臟均未見(jiàn)高密度影;第7~10周誘石組大鼠腎臟可見(jiàn)白色顆粒樣高密度影。第1~10周,誘石組大鼠腎臟高密度影檢出4只(13.3%),對(duì)照組未檢出;截至第10周末,兩組大鼠腎臟高密度影檢出率比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.056)。病理組織學(xué)檢查結(jié)果:第1~10周,對(duì)照組大鼠腎臟內(nèi)無(wú)晶體沉積。第4周及第6~10周誘石組大鼠腎臟可見(jiàn)灰白色晶體。第1~10周,誘石組大鼠腎臟晶體陽(yáng)性11只(36.7%),對(duì)照組均為陰性;截至第10周末,誘石組大鼠腎臟晶體陽(yáng)性率高于對(duì)照組(P<0.001)。結(jié)論 納米細(xì)菌在第4周開(kāi)始誘導(dǎo)大鼠腎臟形成結(jié)晶,這個(gè)病理過(guò)程在第6~10周持續(xù)存在,Mirco-CT檢查可見(jiàn)第7~10周形成高密度結(jié)石樣物質(zhì),納米細(xì)菌可能通過(guò)早期損傷腎臟誘導(dǎo)結(jié)晶形成并進(jìn)一步產(chǎn)生結(jié)石。
腎結(jié)石;納米細(xì)菌;模型,動(dòng)物;體層攝影術(shù),螺旋計(jì)算機(jī)
褚浩,王勤章,吳雙,等.納米細(xì)菌大鼠腎結(jié)石模型腎臟結(jié)石形成時(shí)間的動(dòng)態(tài)研究[J].中國(guó)全科醫(yī)學(xué),2017,20(21):2613-2618.[www.chinagp.net]
CHU H,WANG Q Z,WU S,et al.Dynamic study on the time of stone formation in rat nephrolithiasis model induced by nanobacteria[J].Chinese General Practice,2017,20(21):2613-2618.
納米細(xì)菌自從被發(fā)現(xiàn)以來(lái),其身影出現(xiàn)于生物體內(nèi)的各種鈣化現(xiàn)象,且一直是泌尿系結(jié)石病因?qū)W的研究熱點(diǎn)。納米細(xì)菌在腎結(jié)石形成中的作用一直備受關(guān)注,國(guó)內(nèi)外相關(guān)學(xué)者常通過(guò)納米細(xì)菌感染動(dòng)物建立相關(guān)腎結(jié)石模型,從血尿生化檢測(cè)、病理切片觀察等方面做出針對(duì)性的探討研究[1-4]。本研究在借鑒上述經(jīng)典方法的同時(shí),參考在骨骼等高密度物質(zhì)研究方面有較廣泛應(yīng)用的微計(jì)算機(jī)斷層掃描技術(shù)(Mirco-CT)這一相對(duì)新穎的技術(shù)手段,首次采用Mirco-CT動(dòng)態(tài)觀察感染納米細(xì)菌的大鼠腎結(jié)石形成情況,同時(shí)進(jìn)行病理組織學(xué)檢查,具體報(bào)道如下。
1.1 實(shí)驗(yàn)材料 SPF級(jí)雄性Wistar大鼠60只,6周齡左右,體質(zhì)量(200±20)g,購(gòu)自新疆醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心〔合格證號(hào)SCXK(新) 2013- 0001〕?;撅暳嫌墒幼哟髮W(xué)實(shí)驗(yàn)動(dòng)物中心提供,多功能光學(xué)顯微鏡及圖像采集系統(tǒng)(BX40,日本Olympus公司)、Mirco-CT(Bruker Company)。
1.2 方法
1.2.1 大鼠腎結(jié)石模型建立及實(shí)驗(yàn)取材 本課題組前期收集上尿路結(jié)石患者的尿液,過(guò)濾處理后進(jìn)行細(xì)胞培養(yǎng),得到的納米細(xì)菌懸液經(jīng)相差顯微鏡、電鏡、鈣染色鑒定[5]。2015年11月—2016年6月,60只SPF級(jí)Wistar大鼠適應(yīng)性飼養(yǎng)1周后,隨機(jī)分為對(duì)照組和誘石組,各30只。對(duì)照組大鼠一次性尾靜脈注射0.9%氯化鈉溶液1.2 ml,誘石組大鼠一次性尾靜脈注射納米細(xì)菌懸液1.2 ml。兩組大鼠注射后第1~10周每周處死3只,收集雙側(cè)腎臟,并置于10%甲醛溶液中固定。
1.2.2 Mirco-CT檢查 將大鼠腎臟固定在放射源和照相機(jī)之間,并保持在可視范圍內(nèi),沿垂直長(zhǎng)軸做0.9°~180.0°旋轉(zhuǎn),并同時(shí)保持在掃描視野范圍內(nèi)。Mirco-CT檢查條件:功率8 W、電壓30 kV,電流300 μA,空間分辨率35 μm,F(xiàn)ilter=0.5 mm AL,觀察大鼠腎臟是否存在高密度影,雙側(cè)腎臟同時(shí)出現(xiàn)高密度影時(shí)僅計(jì)1次,統(tǒng)計(jì)第1~10周兩組大鼠檢測(cè)到的腎臟高密度影情況。
本研究背景:
納米細(xì)菌廣泛存在于人體內(nèi),是一種具有超微結(jié)構(gòu)的生物,可以在人體鈣化組織器官或體液中檢測(cè)到,并被認(rèn)為與多種鈣化性疾病有關(guān),具體到納米細(xì)菌與泌尿系結(jié)石的關(guān)聯(lián),從納米細(xì)菌對(duì)腎小管上皮細(xì)胞水平的影響到納米細(xì)菌感染大鼠動(dòng)物模型的構(gòu)建,國(guó)內(nèi)外學(xué)者均做出一定的探索,觀察到納米細(xì)菌與結(jié)石的形成具有一定的相關(guān)性,這種改變可能與納米細(xì)菌的細(xì)胞毒性作用或礦化能力有關(guān),具體機(jī)制有待進(jìn)一步研究。
1.2.3 病理組織學(xué)檢查 于10%甲醛溶液中取出腎臟常規(guī)脫水,石蠟包埋制片后固定于載玻片上,HE染色,光學(xué)顯微鏡下觀察晶體的形成情況,參考文獻(xiàn)[6]提供的腎臟判定標(biāo)準(zhǔn)進(jìn)行統(tǒng)計(jì),0級(jí)為陰性結(jié)果,Ⅰ~Ⅳ級(jí)為陽(yáng)性結(jié)果,雙側(cè)腎臟同時(shí)出現(xiàn)陽(yáng)性結(jié)果時(shí),僅計(jì)1次,統(tǒng)計(jì)第1~10周兩組大鼠腎臟晶體陽(yáng)性情況。
1.3 統(tǒng)計(jì)學(xué)方法 采用SPSS 17.0統(tǒng)計(jì)學(xué)軟件進(jìn)行數(shù)據(jù)分析。計(jì)數(shù)資料比較采用Fisher確切概率法。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
2.1 Mirco-CT檢查結(jié)果 第1~10周,對(duì)照組大鼠腎臟大體輪廓清晰可見(jiàn),邊緣無(wú)毛糙;三視圖圖像中腎皮質(zhì)、腎髓質(zhì)及腎盂均未見(jiàn)高密度影,見(jiàn)圖1。誘石組大鼠第7周起腎臟可見(jiàn)白色顆粒樣高密度影分布于腎實(shí)質(zhì)中,三視圖圖像可見(jiàn)高亮點(diǎn)狀影隨機(jī)分布于腎實(shí)質(zhì)的各截面中,見(jiàn)圖2。第1~10周,誘石組大鼠腎臟高密度影檢出4只(13.3%),對(duì)照組未檢出(見(jiàn)表1);截至第10周末,兩組大鼠腎臟高密度影檢出率比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.056)。
2.2 病理組織學(xué)檢查結(jié)果 第1~10周,對(duì)照組大鼠腎臟內(nèi)無(wú)晶體沉積,未見(jiàn)明顯病理學(xué)改變。誘石組大鼠第4周起腎臟石蠟切片可見(jiàn)灰白色晶體,主要分布于遠(yuǎn)曲小管、近曲小管及部分腎小球,周圍形態(tài)不規(guī)則,晶體多呈零星散在分布,少量晶體呈互相連接成片或成堆分布,見(jiàn)圖3。第1~10周,誘石組大鼠腎臟晶體陽(yáng)性11只(36.7%),對(duì)照組均為陽(yáng)性(見(jiàn)表2);截至第10周末,誘石組大鼠腎臟晶體陽(yáng)性率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P<0.001)。
納米細(xì)菌是由Kajander發(fā)現(xiàn)能形成磷灰石碳酸鹽晶體的特殊生物,在人體許多器官組織及體液中廣泛存在[7-8],參與體內(nèi)諸多病理性鈣化過(guò)程[1]。GARCA CUERPO等[2]應(yīng)用培養(yǎng)的納米細(xì)菌經(jīng)皮腎臟穿刺注射,初步建立了納米細(xì)菌致大鼠腎結(jié)石動(dòng)物模型,解剖大鼠后發(fā)現(xiàn)其腎小管可見(jiàn)廣泛鈣化,因此提出納米細(xì)菌作為結(jié)石核心的設(shè)想。胡衛(wèi)國(guó)等[3]、粟宏偉等[4]參照SHIEKH等[9]實(shí)驗(yàn)大鼠尾靜脈注射納米細(xì)菌,8周后發(fā)現(xiàn)實(shí)驗(yàn)組大鼠腎小管管腔內(nèi)不規(guī)則片狀高亮晶體,主要分布于遠(yuǎn)曲小管和近曲小管,進(jìn)一步進(jìn)行結(jié)晶分級(jí)計(jì)數(shù),發(fā)現(xiàn)與經(jīng)典的乙二醇氯化銨誘導(dǎo)腎結(jié)石模型相比,納米細(xì)菌誘導(dǎo)的大鼠腎結(jié)石模型晶體數(shù)量少,腎盂等集合系統(tǒng)內(nèi)晶體或結(jié)石形成極少,腎小管內(nèi)的晶體少有連接成片或成堆。但上述研究對(duì)于納米細(xì)菌的結(jié)石形成過(guò)程僅進(jìn)行了總結(jié)性的報(bào)道,缺乏動(dòng)態(tài)的觀察描述,因此,本研究建立納米細(xì)菌大鼠腎結(jié)石模型,以造模后10周為時(shí)間截點(diǎn),觀察大鼠各周腎臟結(jié)石形成情況,并引入近年來(lái)發(fā)展較快的Mirco-CT,實(shí)現(xiàn)了對(duì)樣本的空間結(jié)構(gòu)、微觀結(jié)構(gòu)和重塑性的三維可視化同步呈現(xiàn),同時(shí)兼有快捷方便、無(wú)創(chuàng)性等特點(diǎn),其在觀察和測(cè)量高密度物質(zhì)方面相對(duì)于傳統(tǒng)的組織切片技術(shù)具有明顯優(yōu)勢(shì)[10-11],因此在分析研究骨骼等高密度物質(zhì)的實(shí)驗(yàn)研究領(lǐng)域得到一定應(yīng)用[12-13]。鑒于結(jié)石是高密度物質(zhì)的物理特性,本實(shí)驗(yàn)首次嘗試?yán)肕irco-CT,定性分析大鼠腎臟結(jié)石形成情況,同時(shí)分析腎臟HE染色結(jié)果,共同探討納米細(xì)菌大鼠腎結(jié)石模型結(jié)石形成的時(shí)間規(guī)律特點(diǎn)。
注:A為腎臟CT圖,腎盂向前:B、C、D依次為矢狀面、橫截面、冠狀面
圖1 對(duì)照組大鼠第7周腎臟Mirco-CT檢查結(jié)果
Figure 1 Results of kidney Mirco-CT examination in control group of rats for the seventh week
注:A為腎臟CT圖,腎盂向前:B、C、D依次為矢狀面、橫截面、冠狀面
圖2 誘石組組大鼠第7周腎臟Mirco-CT檢查結(jié)果
表2 兩組大鼠各周腎臟晶體陽(yáng)性情況(只)
注:A為對(duì)照組大鼠腎臟,B為誘石組大鼠腎臟
圖3 兩組大鼠第6周腎臟病理組織圖(HE染色,×200)
Figure 3 Pathological structure of kidney in two groups of rats for the sixth week
本實(shí)驗(yàn)發(fā)現(xiàn)Mirco-CT檢查和病理組織學(xué)檢查結(jié)果提示誘石組的結(jié)石檢出率較對(duì)照組高,但是Mirco-CT檢查結(jié)果無(wú)統(tǒng)計(jì)學(xué)差異,可能與腎結(jié)石程度較輕、個(gè)別標(biāo)本未被Mirco-CT檢查檢出而呈假陰性有關(guān)。Mirco-CT檢查示,第7~10周誘石組大鼠均有腎臟高密度影檢出,對(duì)照組大鼠未檢出。病理組織學(xué)檢查結(jié)果顯示,第4周及第6~10周誘石組大鼠腎小管內(nèi)發(fā)現(xiàn)晶體,且主要分布于遠(yuǎn)曲小管和近曲小管,對(duì)照組大鼠未見(jiàn)晶體。相關(guān)研究顯示,乙二醇誘導(dǎo)腎結(jié)石周期普遍為4周左右[14-15],而納米細(xì)菌誘導(dǎo)大鼠結(jié)石造模周期較長(zhǎng),這可能與納米細(xì)菌的生物學(xué)代謝特點(diǎn)有關(guān)。納米細(xì)菌只能在細(xì)胞培養(yǎng)基上生長(zhǎng),且生長(zhǎng)緩慢,倍增時(shí)間平均為3~5 d,細(xì)胞培養(yǎng)常需要4~6周以上才能出現(xiàn)肉眼可見(jiàn)白色沉淀[7,16]。ZHOU等[17]經(jīng)大鼠尿道將納米細(xì)菌逆行感染前列腺,然后分別在不同時(shí)間點(diǎn)處死大鼠,收集標(biāo)本檢測(cè)感染率達(dá)95%,對(duì)照組無(wú)感染大鼠,組間差異明顯;光鏡下大鼠感染4周后前列腺均表現(xiàn)為慢性炎癥;透射電子顯微鏡下胞質(zhì)內(nèi)可見(jiàn)聚集的球狀或桿狀納米細(xì)菌存在,周圍出現(xiàn)空泡狀、透明樣結(jié)構(gòu),本研究觀察到的腎臟病理?yè)p害與之相印證,同時(shí)體外細(xì)胞實(shí)驗(yàn)[18-19]發(fā)現(xiàn)納米細(xì)菌不僅通過(guò)損傷腎小管上皮細(xì)胞誘導(dǎo)大鼠腎小管內(nèi)產(chǎn)生含鈣結(jié)晶,而且損傷細(xì)胞后,細(xì)胞的晶體黏附性明顯增強(qiáng),隨損傷作用時(shí)間的延長(zhǎng),細(xì)胞與晶體的黏附量也會(huì)相應(yīng)增加[20],上述過(guò)程相互協(xié)同、共同促進(jìn)腎結(jié)石的形成。鈣化和結(jié)石形成均離不開(kāi)納米細(xì)菌獨(dú)特的生物礦化作用,并且均是以納米細(xì)菌為核心,通過(guò)不斷結(jié)合鈣離子,逐漸在其周圍聚集,形成沉淀并發(fā)展為鈣化、結(jié)石等[21]。
與納米細(xì)菌腎結(jié)石模型相比,傳統(tǒng)的乙二醇誘導(dǎo)草酸鈣大鼠結(jié)石模型建立后,能較快的觀察到結(jié)石出現(xiàn),雖然二者的原發(fā)機(jī)制可能不盡相同,但是均出現(xiàn)腎臟損害及結(jié)晶形成的現(xiàn)象,這就有利的佐證了納米細(xì)菌對(duì)腎結(jié)石形成的促進(jìn)作用,同時(shí),納米細(xì)菌的鈣化作用受自身生理?xiàng)l件的影響較大,正因?yàn)槿绱耍{米細(xì)菌誘導(dǎo)的成石模型在某方面可能更接近人類腎結(jié)石疾病的代謝過(guò)程,對(duì)相關(guān)研究也是一個(gè)有益的探索及嘗試。
綜上所述,納米細(xì)菌在第4周開(kāi)始誘導(dǎo)大鼠腎臟形成結(jié)晶,這個(gè)病理過(guò)程在第6~10周持續(xù)存在,Mirco-CT檢查可見(jiàn)第7~10周形成高密度結(jié)石樣物質(zhì)。納米細(xì)菌在利用自身礦化特性誘導(dǎo)結(jié)晶形成的同時(shí)損傷腎小管等腎臟結(jié)構(gòu),上述病理過(guò)程相互作用,互相協(xié)同,進(jìn)一步導(dǎo)致腎臟結(jié)構(gòu)的變化,最終導(dǎo)致結(jié)石的形成。作為腎結(jié)石病因?qū)W的一個(gè)研究方向,全面闡明納米細(xì)菌在整個(gè)疾病變化中的機(jī)制還需要更廣泛、更深入的研究,例如在細(xì)胞學(xué)水平上進(jìn)行更多的時(shí)間點(diǎn)觀察并檢測(cè)更多相關(guān)蛋白及其他指標(biāo)的表達(dá),了解納米細(xì)菌對(duì)相關(guān)指標(biāo)的影響情況,為腎結(jié)石的病因研究及診治預(yù)防做進(jìn)一步探索。
作者貢獻(xiàn):褚浩、王勤章、錢(qián)彪進(jìn)行文章的構(gòu)思與設(shè)計(jì),研究的實(shí)施與可行性分析,撰寫(xiě)論文,論文修訂;褚浩、吳雙、錢(qián)成、王議鶴進(jìn)行數(shù)據(jù)收集與整理;褚浩進(jìn)行統(tǒng)計(jì)學(xué)處理,結(jié)果分析與解釋,負(fù)責(zé)文章的質(zhì)量控制及審校;褚浩、王勤章、錢(qián)彪對(duì)文章整體負(fù)責(zé),監(jiān)督管理。
本文無(wú)利益沖突。
本研究不足之處:
目前國(guó)內(nèi)納米細(xì)菌均是通過(guò)收集人體標(biāo)本進(jìn)行細(xì)胞培養(yǎng)獲得,培養(yǎng)周期至少6~8周,并且每個(gè)培養(yǎng)瓶獲得納米細(xì)菌的數(shù)量有限,而實(shí)驗(yàn)造模要求納米細(xì)菌提取后要達(dá)到一定濃度,這就使最終納米細(xì)菌的數(shù)量受到限制,能被其感染的大鼠數(shù)量相應(yīng)受到限制,樣本量較小,整體陽(yáng)性率不高,同時(shí)微計(jì)算機(jī)斷層掃描技術(shù)(Mirco-CT)的應(yīng)用尚顯粗糙,有待進(jìn)一步改善和研究。
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(本文編輯:毛亞敏)
Dynamic Study on the Time of Stone Formation in Rat Nephrolithiasis Model Induced by Nanobacteria
CHUHao,WANGQin-zhang,WUShuang,QIANCheng,WANGYi-he,QIANBiao*
DepartmentofUrologySurgery,theFirstAffiliatedHospitalofMedicalSchoolofShiheziUniversity,Shihezi832000,China
Objective To dynamically observe the stone formation in rat nephrolithiasis model induced by nanobacteria by using micro computed tomography(Mirco-CT) examination and histopathological examination.Methods From November 2015 to June 2016,60 SPF class Wistar rats were adaptively fed for one week.Then the rats were randomly divided into two groups:control group and induced stone group,30 rats in each group.The rats in the control group were injected with 1.2 ml of 0.9% sodium chloride solution via the tail vein at one time,the rats in the induced stone group were injected with 1.2 ml of nanobacterial suspension at one time.Three rats in each group were sacrificed every week from the 1st to 10th week after injection,bilateral renal tissues were collected to be tested.The kidneys of the two groups of rats were examined by Mirco-CT,the detected high density shadow in the kidneys of the two groups of rats at the 1st to 10th week was statistically analyzed.The crystal formation in the renal tissues of the two groups of rats was observed under light microscope,and the positive condition in the renal of the two groups of rats at the 1st to 10th week was statistically analyzed.Results Mirco-CT scan results showed that there was no high density shadow in the kidneys of rats in the control group from the 1st to 10th week,while there were white high density granules in the kidneys of the rats in induced stone group from the 7th to 10th week.From the 1st to 10th week,high density shadow was detected in the renal tissues of 4 rats(13.3%) in induced stone group,while not in the control group.At the end of the 10th week,there was no significant difference in the detection rate of high density shadow in renal tissues between the two groups(P=0.056).Histopathological findings showed that there were no crystal deposits in the kidneys of the rats in the control group from the 1st to 10th week.At the 4th week and the 6th to 10th week,greyish white crystals were detected in the renal tissues of the rats in the induced stone group.From the 1st to 10th week,crystals were detected in the renal tissues of 11 rats(36.7%) in induced group,but not in the control group.At the end of the 10th week,the crystal detection rate by paraffin section in the renal tissues of rats in induced stone group was higher than that in the control group,the difference was statistically significant(P<0.001).Conclusion Nanobacteria begin to induce the formation of crystals in the kidneys of the rats at the 4th week.The pathological process persists at the 6th to 10th week.Finally,the Mirco-CT examination shows the formation of high density stone like substance at the 7th to 10th week.Nanobacteria may induce the formation of crystals by early renal injury and further produce stones.
Kidney calculi;Nanobacteria;Models,animal;Tomography,spiral computed
國(guó)家自然科學(xué)基金資助項(xiàng)目(81460140)
R 692.4
A
10.3969/j.issn.1007-9572.2017.21.011
2017-02-23;
2017-06-06)
832000新疆石河子市,石河子大學(xué)醫(yī)學(xué)院第一附屬醫(yī)院泌尿外科
*通信作者:錢(qián)彪,副教授,副主任醫(yī)師;E-mail:qb2003_2000@163.com
*Correspondingauthor:QIANBiao,Associateprofessor,Associatechiefphysician;E-mail:qb2003_2000@163.com