本文選題：卡介苗 + 膀胱灌注��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景 膀胱癌是泌尿系統(tǒng)最常見(jiàn)的惡性腫瘤之一,發(fā)病率較高。膀胱癌包括尿路上皮癌、鱗狀細(xì)胞癌和腺細(xì)胞癌,其中尿路上皮癌最為常見(jiàn),雖然理論上經(jīng)尿道膀胱腫瘤電切術(shù)可完全切除非肌層浸潤(rùn)性膀胱癌,但術(shù)后復(fù)發(fā)率可高達(dá)67%。因此中國(guó)、歐洲及美國(guó)的泌尿外科指南建議所有非肌層浸潤(rùn)性膀胱癌患者術(shù)后均應(yīng)進(jìn)行輔助性膀胱灌注治療�？ń槊�(Bacillus Calmette-Guerin, BCG)是膀胱灌注常用的一種生物制劑,為一種減毒活菌,具有一定的抗原性、致敏性和殘余毒性。經(jīng)過(guò)多年的實(shí)踐,BCG己被公認(rèn)為最有效的預(yù)防膀胱腫瘤復(fù)發(fā)的免疫制劑,適用于表淺的、非肌層浸潤(rùn)性膀胱腫瘤的術(shù)后灌注治療。 BCG膀胱灌注雖然療效確切,但其不良反應(yīng)發(fā)生率較高,限制了其臨床應(yīng)用。且有研究報(bào)道,30～50%的膀胱腫瘤病人BCG灌注失敗,因無(wú)法耐受副作用、對(duì)BCG治療反應(yīng)差、腫瘤進(jìn)展或復(fù)發(fā)等,最終停用BCG灌注治療。增加BCG的灌注劑量是增強(qiáng)BCG治療反應(yīng)的最直接方法,但矛盾的是BCG灌注的劑量越大,雖然抗腫瘤效果明顯,但副作用隨之增加,如何提高BCG的療效同時(shí)降低其不良反應(yīng),成為研究的熱點(diǎn)。 由于人周期性的排尿,藥物灌注后很快隨尿液排出體外,其與膀胱粘膜的作用時(shí)間較短,通常只有1～2小時(shí)。藥物膀胱內(nèi)灌注后,在較短的時(shí)間段內(nèi),病人對(duì)膀胱內(nèi)藥物的反應(yīng)個(gè)體差異較大,部分病人甚至沒(méi)有反應(yīng)。膀胱內(nèi)藥物灌注的效果主要與膀胱內(nèi)藥物濃度及持續(xù)時(shí)間相關(guān),而非藥物灌注劑量。因此,BCG灌注后在膀胱內(nèi)的持續(xù)作用時(shí)間對(duì)治療效果至關(guān)重要。 基于殼聚糖(CS)及p-甘油磷酸鈉(β-glycerophosphate, GP)的溫敏凝膠是一種生物材料,在室溫條件下,CS和GP的混合溶液呈液態(tài),加熱至37℃體溫條件下,即轉(zhuǎn)變?yōu)镃S/GP凝膠。CS凝膠在藥物載體、細(xì)胞包裹及組織工程等方面得到廣泛應(yīng)用,是優(yōu)良且極具發(fā)展前途的醫(yī)用緩釋體系。因此我們?cè)O(shè)想,CS/GP溫敏凝膠可以裝載BCG進(jìn)行膀胱灌注,以期延長(zhǎng)BCG在膀胱內(nèi)的滯留時(shí)間,提高BCG的療效。 CS/GP溫敏凝膠載體進(jìn)入膀胱后依然會(huì)隨尿液排出體外。我們?cè)O(shè)想利用磁性顆粒及外加磁場(chǎng)防止凝膠載體隨尿液排出。Fe304磁性納米粒(Fe3O4magnetic nanoparticle, Fe3O4-MNP)是一種納米級(jí)鐵氧體,具有磁響應(yīng)性好,在液體中可均勻分散,且在生物體內(nèi)可被降解,無(wú)毒副作用等特性,作為磁性材料在生物領(lǐng)域中得到廣泛應(yīng)用。因此我們將Fe3O4-MNP包裝進(jìn)入CS/GP溫敏凝膠內(nèi),在外加磁場(chǎng)條件下,利用Fe3O4-MNP所提供的磁牽引力,實(shí)現(xiàn)凝膠載體在膀胱內(nèi)的貼壁粘附。 研究目的 本課題中我們以CS/GP凝膠為基質(zhì),設(shè)計(jì)了一種搭載BCG、Fe3O4-MNP的載體復(fù)合物(Fe3O4-BCG-CS/GP),在常溫條件下為液體,進(jìn)入體內(nèi)后可轉(zhuǎn)變?yōu)槟z,用以進(jìn)行BCG的膀胱灌注,以期延長(zhǎng)BCG在膀胱內(nèi)的作用時(shí)間,并最終增強(qiáng)BCG的抗膀胱腫瘤作用。 方法 1. Fe3O4-BCG-CS/GP凝膠載體系統(tǒng)的構(gòu)建、表征檢測(cè)及體內(nèi)應(yīng)用可行性評(píng)估 1.1Fe3O4-BCG-CS/GP載體復(fù)合物的制備 根據(jù)已有文獻(xiàn)報(bào)道及我們的前期實(shí)驗(yàn),制備方法如下： 1)將脫乙酰度為95%的CS溶于0.1mol/L的稀鹽酸中； 2)室溫下持續(xù)充分?jǐn)嚢?小時(shí)； 3)將CS溶液中不溶性顆粒過(guò)濾； 4)將p-GP粉末溶于蒸餾水中； 5)將CS溶液及β-GP溶液在4℃C冰箱中冷藏10分鐘； 6)持續(xù)攪拌的狀態(tài)下將p-GP溶液逐滴加入CS溶液中,直至形成均一的溶液； 7)然后將一定量的BCG及Fe3O4-MNP加入混合溶液中,采用機(jī)械攪拌、超聲分散的方法混合均勻； 1.2CS/GP溶液及Fe3O4-BCG-CS/GP復(fù)合物溶液的體外凝膠化時(shí)間測(cè)定 不同濃度配比的CS/GP溶液及Fe3O4-BCG-CS/GP載體復(fù)合物凝膠化時(shí)間的測(cè)定按照如下經(jīng)典方法進(jìn)行： 將溶液加入離心管中；37℃水浴鍋中加熱；按照一定的時(shí)間間隔,從水浴鍋中取出離心管,倒置觀察；倒置30s樣品不流動(dòng),即可認(rèn)為凝膠化完成；記錄不同CS和GP配比的CS/GP溶液凝膠化時(shí)間,Fe3O4-BCG-CS/GP載體復(fù)合物溶液凝膠化時(shí)間同理測(cè)定。 1.3大鼠體內(nèi)凝膠化時(shí)間測(cè)定及冰凍切片檢查 利用大鼠測(cè)定體內(nèi)凝膠化時(shí)間。灌注前,進(jìn)行腹腔注射麻醉,取3Fr硬膜外導(dǎo)管自制大鼠導(dǎo)尿管。用空針抽出大鼠膀胱內(nèi)尿液。將0.1ml Fe3O4-BCG-CS/GP載體復(fù)合物溶液注入大鼠膀胱內(nèi)。根據(jù)步驟2中測(cè)定的凝膠化時(shí)間,處死大鼠,取出膀胱,觀察凝膠化完成情況。然后對(duì)大鼠膀胱進(jìn)行冰凍切片檢查,觀察復(fù)合物凝膠后的形態(tài)。 1.4數(shù)碼顯微鏡及掃描電鏡檢查 我們采用數(shù)碼顯微鏡及掃描電鏡來(lái)觀察Fe3O4-BCG-CS/GP復(fù)合物凝膠的表面形態(tài)。Fe3O4-BCG-CS/GP復(fù)合物溶液在37℃水浴中完成凝膠化。取樣品少量冷凍干燥。放置于數(shù)碼顯微鏡下觀察攝片。噴鍍貴金屬銀,增強(qiáng)導(dǎo)電性,處理完成后在掃描電鏡下觀察復(fù)合物凝膠的表面形態(tài)。 1.5Fe3O4-BCG-CS/GP復(fù)合物凝膠大鼠膀胱內(nèi)滯留時(shí)間測(cè)定 雌性’Wistar大鼠36只用以測(cè)定Fe3O4-BCG-CS/GP復(fù)合物凝膠在膀胱內(nèi)的滯留時(shí)間。大鼠膀胱灌注前2小時(shí)禁飲。麻醉后進(jìn)行膀胱灌注,所有大鼠處于4kG外加磁場(chǎng)中飼養(yǎng)。灌注后每4小時(shí)處死大鼠3只；取膀胱標(biāo)本立即進(jìn)行冰凍切片檢查,HE染色觀察膀胱內(nèi)殘留凝膠形態(tài),抗酸染色及復(fù)染HE染色觀察凝膠中剩余BCG的形態(tài)。 2. Fe3O4-BCG-CS/GP凝膠載體系統(tǒng)抗膀胱腫瘤作用的研究 2.1實(shí)驗(yàn)設(shè)計(jì) 本部分實(shí)驗(yàn)使用24只雌性Wistar大鼠。根據(jù)以往成熟的實(shí)驗(yàn)設(shè)計(jì),采用BBN誘發(fā)大鼠膀胱腫瘤原位模型后進(jìn)行抗腫瘤實(shí)驗(yàn)。BBN加入每日飲水中,濃度為0.05%,共喂養(yǎng)8周。10周后,24只大鼠根據(jù)灌注藥物的不同隨機(jī)分為4組： 第一組(Group1):為對(duì)照組,給予0.1ml的PBS灌注； 第二組(Group2):給予1mg/0.1ml的BCG進(jìn)行膀胱灌注； 第三組(Group3):CS/GP溶液灌注組,每次灌注0.1ml； 第四組(Group4):給予Fe3O4-BCG-CS/GP復(fù)合物溶液0.1ml,含1mg BCG。四組大鼠每周灌注一次,共6次。實(shí)驗(yàn)的1-8周為腫瘤誘導(dǎo)期,11～16周為治療期。四組大鼠均飼養(yǎng)于強(qiáng)度為4kG的磁場(chǎng)環(huán)境中。 2.2抗腫瘤效果驗(yàn)證 20周后麻醉下處死所有大鼠,收集大鼠膀胱組織觀察。詳細(xì)記錄每只大鼠的腫瘤數(shù)目及每個(gè)腫瘤的體積,計(jì)算每組大鼠的腫瘤發(fā)生率及每只大鼠的平均腫瘤體積。直徑超過(guò)0.5mm的腫物擬定為腫瘤,列入統(tǒng)計(jì)中。腫瘤體積按如下公式計(jì)算： V (mm3)=1/2XaXb2(a是腫瘤的最大直徑,b為腫瘤的最短直徑)。 2.3尿液細(xì)胞因子分析 膀胱灌注后,將4組大鼠轉(zhuǎn)移至代謝籠內(nèi)收集尿液。第一次膀胱灌注后24小時(shí)收集尿液,每日收集1次,共6次。按實(shí)驗(yàn)計(jì)劃,在處死大鼠前收集尿液1次。每次收集的時(shí)間約2小時(shí)。收集尿液的離心管內(nèi)含蛋白酶抑制劑,收集時(shí)放置于冰塊上。收集完成后離心、儲(chǔ)存于-80℃冰箱中待檢。本實(shí)驗(yàn)采用雙抗體夾心ELISA法來(lái)檢測(cè)大鼠尿液中IL-2和IFN-γ的濃度,描繪出其變化曲線。通過(guò)計(jì)算曲線下面積(area under the curve, AUC)來(lái)估算大鼠接受單次膀胱灌注后尿液中細(xì)胞因子分泌的總量,據(jù)此評(píng)估大鼠膀胱內(nèi)免疫反應(yīng)的強(qiáng)弱。 2.4免疫組化檢查 大鼠膀胱灌注BCG后引起膀胱內(nèi)CD4+的淋巴細(xì)胞浸潤(rùn),其在BCG抗膀胱腫瘤的作用機(jī)制中起重要作用。處死所有大鼠后,收集大鼠膀胱組織進(jìn)行免疫組化檢查。我們采用CD4抗體免疫組化染色的方法檢測(cè)膀胱粘膜中浸潤(rùn)的CD4+淋巴細(xì)胞。 2.5統(tǒng)計(jì)分析 所有數(shù)據(jù)讀取等檢測(cè)步驟重復(fù)進(jìn)行三遍,指標(biāo)進(jìn)行統(tǒng)計(jì)學(xué)處理,采用Prism5統(tǒng)計(jì)軟件進(jìn)行分析,計(jì)量數(shù)值以x±s表示,根據(jù)需要選用配對(duì)資料的t檢驗(yàn)或單因素方差分析進(jìn)行統(tǒng)計(jì)分析,選用雙側(cè)檢驗(yàn)。P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1. Fe3O4-BCG-CS/GP凝膠載體系統(tǒng)的構(gòu)建、表征檢測(cè)及體內(nèi)應(yīng)用可行性評(píng)估 實(shí)驗(yàn)所制備的CS/GP溶液無(wú)色半透明狀,性質(zhì)均一,在體外加熱至37℃C的條件下,CS/GP溶液成功完成凝膠化,凝膠化后溶液失去流動(dòng)性。傾倒離心管,凝膠固定不流動(dòng)。Fe3O4-BCG-CS/GP復(fù)合物成功完成凝膠化,樣品呈灰黑色,性質(zhì)均一。實(shí)驗(yàn)發(fā)現(xiàn),CS及GP的濃度皆可影響凝膠化時(shí)間。由于復(fù)合物凝膠中Fe3O4-MNP的存在,凝膠保留了一定的磁響應(yīng)性。經(jīng)冰凍切片、HE染色后觀察,Fe3O4-BCG-CS/GP載體復(fù)合物亦在大鼠膀胱內(nèi)完成凝膠化。由于CS為有機(jī)物,冰凍切片法較好地保留了復(fù)合物凝膠在膀胱內(nèi)的形態(tài),顯示Fe3O4-BCG-CS/GP復(fù)合物凝膠載體呈網(wǎng)格狀的微結(jié)構(gòu),大小、直徑不一。數(shù)碼顯微鏡及電鏡下可觀察到復(fù)合物凝膠的表面同樣呈現(xiàn)大小不同的網(wǎng)格狀結(jié)構(gòu),與冰凍切片所示結(jié)構(gòu)相似。在磁場(chǎng)作用下,復(fù)合物凝膠在膀胱內(nèi)貼附到膀胱壁表面。隨著復(fù)合物凝膠在膀胱內(nèi)作用時(shí)間的延長(zhǎng),其在粘膜表面的降解增多、隨尿液排出的損耗增大以及尿液的溶蝕作用,其濃度和密度也逐漸降低,凝膠網(wǎng)格溶脹擴(kuò)大,網(wǎng)格壁變薄。經(jīng)過(guò)48小時(shí),我們?nèi)钥稍诎螂變?nèi)檢測(cè)到少量凝膠存留,因此我們可以認(rèn)為,磁性凝膠作為BCG載體,可以在外加磁場(chǎng)的作用下,在膀胱內(nèi)滯留時(shí)間長(zhǎng)達(dá)48個(gè)小時(shí)。 2. Fe3O4-BCG-CS/GP凝膠載體系統(tǒng)抗膀胱腫瘤作用的研究 在抗大鼠膀胱腫瘤的研究中,大鼠灌注Fe3O4-BCG-CS/GP復(fù)合物后,所有24只大鼠均按計(jì)劃完成20周的實(shí)驗(yàn)過(guò)程。在第20周末,處死大鼠,取大鼠膀胱組織進(jìn)行檢查。Fe3O4-BCG-CS/GP復(fù)合物與普通BCG都顯示出明顯抗腫瘤誘導(dǎo)作用。Fe3O4-BCG-CS/GP復(fù)合物組大鼠的平均腫瘤體積較BCG組大鼠明顯減小,但在腫瘤數(shù)目方面的差異沒(méi)有統(tǒng)計(jì)學(xué)意義。Fe3O4-BCG-CS/GP復(fù)合物灌注后大鼠尿液中IL-2及IFN-γ濃度較普通BCG組明顯升高,且分泌總量較大。通過(guò)CD4染色,Fe3O4-BCG-CS/GP復(fù)合物灌注的大鼠膀胱粘膜下有更多CD4+淋巴細(xì)胞浸潤(rùn)。 結(jié)論 本實(shí)驗(yàn)中成功制備了以CS/GP溫敏凝膠為載體、以Fe3O4-MNP為磁性牽引物、搭載BCG的Fe3O4-BCG-CS/GP凝膠載體系統(tǒng),經(jīng)驗(yàn)證可在體內(nèi)、體外成功完成凝膠化,保留了較好的磁響應(yīng)性。凝膠載體呈網(wǎng)格狀,BCG等搭載物在凝膠載體中相對(duì)獨(dú)立,利于BCG的釋放。Fe3O4-BCG-CS/GP凝膠載體系統(tǒng)在外加磁場(chǎng)作用下,可在大鼠膀胱內(nèi)延長(zhǎng)BCG的作用時(shí)間達(dá)48小時(shí)；動(dòng)物實(shí)驗(yàn)顯示,與普通BCG相比,同等劑量條件下Fe3O4-BCG-CS/GP凝膠載體所搭載的BCG可刺激膀胱分泌更多細(xì)胞因子,募集大量炎性細(xì)胞浸潤(rùn),在膀胱內(nèi)產(chǎn)生更強(qiáng)的粘膜免疫反應(yīng),其抗膀胱腫瘤作用優(yōu)于普通BCG。我們認(rèn)為以CS/GP溫敏凝膠為載體搭載Fe3O4-MNP、BCG后構(gòu)建的Fe3O4-BCG-CS/GP凝膠載體系統(tǒng),增強(qiáng)了BCG的免疫活性及抗腫瘤效應(yīng),為BCG的應(yīng)用提供了一種新的思路,有望成為用于膀胱癌術(shù)后灌注預(yù)防膀胱癌復(fù)發(fā)的更好的免疫制劑。
[Abstract]:Research background
Bladder cancer is one of the most common malignant tumor in urinary system, high incidence of bladder cancer. Including urothelial carcinoma, squamous cell carcinoma and adenocarcinoma, including urothelial carcinoma is the most common, although the theory of transurethral resection of bladder tumor complete resection of non muscle invasive bladder cancer, but the operation the recurrence rate can be as high as 67%. so Chinese, Department of Urology guide to Europe and the United States suggest that all non muscle invasive bladder cancer patients should be adjuvant intravesical therapy. BCG (Bacillus Calmette-Guerin BCG) is a biological agent used for bladder perfusion, a live attenuated bacteria, has a certain the antigenicity and allergenicity and toxicity. After years of practice, BCG has been recognized as immune agents in the prevention of recurrence of bladder tumor is the most effective, suitable for superficial, non muscle invasive bladder cancer after reperfusion therapy.
Intravesical instillation of BCG although the exact curative effect, but the adverse reaction rate is high, limiting its clinical application. And studies have reported that 30 to 50% of patients with bladder cancer BCG perfusion failure due to intolerable side effects, poor response to BCG treatment, tumor progression or recurrence, eventually stopping BCG perfusion. Increase perfusion dose BCG is the most direct method to enhance the response to BCG treatment, but the contradiction is more BCG perfusion dose, although the anti-tumor effect is obvious, but the side effect increases, how to improve the efficacy of BCG and reduce the adverse reaction, has become a hot research topic.
Due to periodic urination, drug perfusion quickly excreted in the urine, the bladder mucosa and the relatively short time, usually only 1~2 hours. Bladder instillation of drug, in a relatively short period of time, differences in response to individual patient on intravesical drug large part of patients did not even bladder reaction. Instillation effect is related with the drug concentration and duration of bladder perfusion, rather than the dose of the drug. Therefore, critical duration in the bladder of the curative effect of BCG after reperfusion.
Based on chitosan (CS) and p- sodium glycerophosphate (beta -glycerophosphate, GP) the temperature sensitive gel is a kind of biological material, under the condition of room temperature, mixed solution of CS and GP is a liquid is heated to 37 DEG C temperature conditions, which is transformed into CS/GP gel.CS gel in the drug carrier, is widely used in fine cell encapsulation and tissue engineering and so on, is an excellent and promising medical delivery system. So we assume that CS/GP thermosensitive gel can be loaded BCG bladder perfusion, in order to prolong the residence time of BCG in the bladder, improve the curative effect of BCG.
CS/GP temperature sensitive gel carrier into the bladder will still be excreted in the urine. We envision the use of magnetic particles and magnetic field to prevent the gel carrier urine.Fe304 magnetic nanoparticles (Fe3O4magnetic nanoparticle Fe3O4-MNP) is a kind of nano ferrite, with good magnetic response, can be uniformly dispersed in the liquid, and in vivo can be degraded, no toxic side effect and other characteristics, as the magnetic material has been widely used in biological field. So we will Fe3O4-MNP packed into CS/GP thermosensitive gel, in the presence of an external magnetic field, using the Fe3O4-MNP provided by the magnetic pull, the gel carrier in the bladder wall adhesion.
research objective
We used CS/GP gel matrix in this paper, the design of a carrier complex equipped with BCG, Fe3O4-MNP (Fe3O4-BCG-CS/GP), is a liquid at room temperature, after entering the body can be transformed into gel, bladder perfusion of BCG to BCG in the bladder to prolong the time, and ultimately enhance the antitumor effect of BCG.
Method
Construction of 1. Fe3O4-BCG-CS/GP gel carrier system, characterization detection and feasibility evaluation of application in vivo
Preparation of 1.1Fe3O4-BCG-CS/GP carrier complex
According to the previous literature and our previous experiments, the preparation methods are as follows:
1) the CS deacetylation of 95% was dissolved in the dilute hydrochloric acid of 0.1mol/L.
2) continuous stirring for 2 hours at room temperature;
3) filtration of insoluble particles in CS solution;
4) p-GP powder was dissolved in distilled water.
5) the CS solution and the beta -GP solution were refrigerated in the 4 C C refrigerator for 10 minutes.
6) in the condition of continuous stirring, the p-GP solution is added to the CS solution by drop by drop, until the homogeneous solution is formed.
7) then a certain amount of BCG and Fe3O4-MNP are added into the mixed solution, and the method of mechanical agitation and ultrasonic dispersion are mixed uniformly.
In vitro gel time determination of 1.2CS/GP solution and Fe3O4-BCG-CS/GP complex solution
The gelation time of CS/GP and Fe3O4-BCG-CS/GP carrier complexes with different concentration ratio was determined according to the following classical methods:
Add the solution to a centrifuge tube; heating in a water bath at a temperature of 37 DEG C; according to certain time intervals, remove the centrifuge tube, inverted from the observation of water bath; inverted 30s sample flow, can complete the gelation that record CS/GP solution; the gelation time of different CS and GP ratio, Fe3O4-BCG-CS/GP carrier complex solution gelation time similarly determined.
Gelation time determination and frozen section examination in 1.3 rats
In vivo determination of gelation time using rats. Before perfusion, intraperitoneal injection anesthesia, epidural catheter 3Fr catheter with self-made rat. Taking the empty needle bladder of rats. The 0.1ml Fe3O4-BCG-CS/GP carrier complex solution was injected into the rat bladder. According to step 2 Determination of gelation time, rats were killed remove the bladder, observe the gelation completion. Then the rat bladder for frozen section examination, observation of compound gel form.
1.4 digital microscope and scanning electron microscope
The surface morphology of.Fe3O4-BCG-CS/GP complex solution, we use the digital microscope and scanning electron microscope to observe Fe3O4-BCG-CS/GP gel gelation completed in 37 DEG C water bath. A small amount of sample freeze drying. Placed on the microscope digital radiography. The precious metal of silver plating, enhanced conductivity, surface morphology after the treatment was observed in complexes in gel under scanning electron microscope.
Determination of the retention time in the bladder of 1.5Fe3O4-BCG-CS/GP complex gel rat
The retention time of female rats' Wistar 36 is used to determination of Fe3O4-BCG-CS/GP compound gel in the bladder. Intravesical instillation of 2 hours before the rats were forbidden to drink. After anesthesia for bladder perfusion, all rats in the 4kG magnetic field in feeding every 4 hours after reperfusion. 3 rats from the bladder; frozen section check immediately, HE staining was observed in bladder residual gel form, acid fast staining and double staining HE staining was used to observe the remaining BCG gel form.
Study on the effect of 2. Fe3O4-BCG-CS/GP gel carrier system on bladder tumor
2.1 experimental design
This part of the experiment used 24 female Wistar rats. According to previous mature experimental design, using the BBN induced rat bladder cancer model after anti tumor experiment.BBN added daily drinking water, the concentration of 0.05%, a total of 8.10 weeks after feeding, 24 rats according to the different perfusion drugs were randomly divided into 4 group:
The first group (Group1): for the control group, the PBS perfusion of 0.1ml was given.
The second group (Group2): the BCG of 1mg/0.1ml was given to the bladder.
Third groups (Group3): CS/GP solution perfusion group, each infusion of 0.1ml;
The fourth group (Group4): Fe3O4-BCG-CS/GP complex solution 0.1ml and 1mg BCG. four groups were perfused once a week for 6 times. The 1-8 week of the experiment was induced by tumor and 11~16 weeks as the treatment period. Four groups of rats were fed in the magnetic field with the intensity of 4kG.
2.2 antitumor effect verification
After 20 weeks, all rats were killed under anesthesia, collection of bladder tissue were observed. The number of records with volume of each rat tumor and each tumor, calculate each rat tumor incidence and the average tumor volume of each rat. The diameter of more than 0.5mm was prepared for the tumor, included in the statistics. The tumor volume calculated by the following formula:
V (mm3) =1/2XaXb2 (a is the largest diameter of the tumor, and B is the shortest diameter of the tumor).
2.3 urine cytokine analysis
After intravesical instillation of 4 groups, will be transferred to metabolism cages of rats to collect urine. First after intravesical instillation of 24 hours urine was collected daily collected 1 times, a total of 6 times. According to the experimental plan, collect urine in 1 rats were killed before. Each collection time of about 2 hours. The centrifugal tube containing urine protease inhibitors, placed on the collection of ice. Collected after centrifugation, stored in the refrigerator -80 C for inspection. This experiment using double antibody sandwich ELISA method to detect the concentration of IL-2 and IFN- y in the urine of rats, describe the change curve. By calculating the area under the curve (area under the curve, AUC) to estimate the amount of cytokine secretion in rats received single bladder perfusion in urine, assessing the immune response in a rat bladder in strength.
2.4 immunohistochemical examination
Intravesical instillation of BCG rats caused by intravesical CD4+ lymphocyte infiltration, play an important role in the antitumor mechanisms of BCG. All rats were sacrificed after collection of bladder tissue of rats by immunohistochemical examination. We used the CD4 antibody of CD4+ lymphocyte infiltration was detected in bladder mucosa.
2.5 statistical analysis
All data read detection steps repeated three times, indexes were statistically analyzed, using Prism5 statistical software, the measurement values were expressed as the X + s, according to the analysis were used for statistical analysis paired t test or ANOVA, using two-sided test.P0.05 the difference was statistically significant.
Result
Construction of 1. Fe3O4-BCG-CS/GP gel carrier system, characterization detection and feasibility evaluation of application in vivo
The experiment was colorless and translucent, CS/GP solution prepared by homogeneous properties, in vitro is heated to 37 DEG C under the condition of C, CS/GP successfully completed the solution gelation solution loses fluidity after gelation. The dumping of the centrifuge tube, fixed flow.Fe3O4-BCG-CS/GP compound gel gel samples were successfully completed, black, uniformity experiments. Found that the concentration of CS and GP can affect the gelation time. Because of the Fe3O4-MNP complex in the gel, the gel retains a certain magnetic response. The frozen section, HE staining, Fe3O4-BCG-CS/GP carrier complex also completed the gelation in the rat bladder. Because CS is organic and frozen section method preserve the complex gel in the bladder shape, showing micro structure, Fe3O4-BCG-CS/GP composite gel carrier was grid size, diameter. A digital microscope and electron microscope can be observed by complex coagulation The glue surface showing the same grid structure in different sizes, similar to the structure shown in frozen sections. Under the action of magnetic field, composite gel in the bladder attached to the bladder wall surface. With the prolongation of time gel in the bladder, the increase in the degradation of the mucosal surface, loss of urine. The urine and dissolution, the concentration and density also decreased, gel swelling grid expansion, grid wall thinning. After 48 hours, we are still in the bladder to detect a small amount of gel retention, so we can think that the magnetic gel as a BCG carrier, in the external magnetic field, the bladder retention time up to 48 hours.
Study on the effect of 2. Fe3O4-BCG-CS/GP gel carrier system on bladder tumor
In the study of rat bladder tumor in rats after perfusion of Fe3O4-BCG-CS/GP complex, all the 24 rats were scheduled to complete the experiment for 20 weeks. At the end of the twentieth weeks, the rats were killed, the bladder tissue of rats were examined with ordinary.Fe3O4-BCG-CS/GP compound BCG showed significant antitumor effects induced by the average tumor.Fe3O4-BCG-CS/GP complex volume group rats compared to BCG rats decreased significantly, but the difference in the number of tumors was not statistically significant.Fe3O4-BCG-CS/GP complex after reperfusion in rat urine IL-2 and IFN- gamma concentration was compared with ordinary BCG group increased, and the secretion of large amount. The CD4 staining of rat bladder mucosa Fe3O4-BCG-CS/GP complexes the perfusion with more CD4+ lymphocytes.
conclusion
In this experiment

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

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