本文關(guān)鍵詞： 微泡 葉酸 超聲 巨噬細(xì)胞 卵巢癌　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分:合成葉酸靶向攜氧載紫杉醇脂質(zhì)微泡并檢測體內(nèi)尋靶能力第一節(jié)制備葉酸靶向攜氧載紫杉醇脂質(zhì)微泡及體內(nèi)靶向能力分析目的:合成葉酸靶向攜氧載紫杉醇脂質(zhì)微泡,探究TOPLMBs的體內(nèi)靶向能力。方法:建立裸鼠人卵巢癌SKOV3腹腔移植瘤模型,觀察動(dòng)物腫瘤模型的生長規(guī)律,獲取腫瘤小體,HE染色,免疫組化法檢測腫瘤組織中葉酸受體(FR)的表達(dá)情況。采用機(jī)械振動(dòng)法分別制備攜氧載紫杉醇脂質(zhì)微泡(OPLMBs)及葉酸靶向攜氧載紫杉醇脂質(zhì)微泡(TOPLMBs),用Di I熒光標(biāo)記。將成功建立卵巢癌模型的裸鼠隨機(jī)分為OPLMBs組、TOPLMBs組和TOPLMBs+葉酸封閉組共3組。分別給予熒光標(biāo)記的TOPLMBs、OPLMBs,在給藥后30 min,24 h和48 h處死裸鼠,獲得腫瘤腹水細(xì)胞及腫瘤小體;葉酸封閉組用葉酸阻斷后加入熒光標(biāo)記的TOPLMBs,給藥后30 min同法獲取腹水細(xì)胞及腫瘤小體,共聚焦顯微鏡觀察熒光分布。取荷瘤裸鼠隨機(jī)分為兩組:TOPLMBs組和OPLMBs組,同法給予熒光標(biāo)記的微泡,獲取腹水細(xì)胞,流式細(xì)胞術(shù)分析腹腔卵巢癌細(xì)胞及巨噬細(xì)胞的熒光攝取情況。結(jié)果:裸鼠卵巢癌腹腔移植瘤模型成瘤率為100%,荷瘤裸鼠平均成瘤時(shí)間為8.2±0.84天,腫瘤組織高度表達(dá)葉酸受體。熒光顯微鏡觀察切片:在給藥后30min、24h及48h,TOPLMBs組的熒光分布明顯多于OPLMBs組,TOPLMBs組在30min熒光表達(dá)最強(qiáng)。在給藥后30min,葉酸封閉組熒光表達(dá)量微弱表達(dá),但與OPLMBs組無明顯差別。流式細(xì)胞術(shù):巨噬細(xì)胞攝取熒光量TOPLMBs組5倍強(qiáng)于OPLMBs組,(P0.05),人卵巢癌SKOV3細(xì)胞攝取熒光量TOPLMs組3倍強(qiáng)于OPLMBs組(P0.05)。結(jié)論:人卵巢癌細(xì)胞株SKOV3腹腔移植瘤模型可成功建立,TOPLMBs與腹腔腫瘤小體及腹水中腫瘤細(xì)胞、腫瘤相關(guān)巨噬細(xì)胞能很好的結(jié)合,TOPLMBs能進(jìn)入腫瘤組織內(nèi)部。TOPLMBs有良好的體內(nèi)尋靶能力。第二節(jié):葉酸靶向攜氧載紫杉醇脂質(zhì)微泡在荷瘤裸鼠的組織分布目的:探討葉酸靶向攜氧載紫杉醇脂質(zhì)微泡的組織分布情況,明確給藥后超聲輻照時(shí)間。方法:將荷瘤裸鼠隨機(jī)分為3組:(a)紫杉醇組(PTX),(b)攜氧載紫杉醇脂質(zhì)微泡組(OPLMBs)(c)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡組(TOPLMBs)。按紫杉醇20 mg/kg的藥量腹腔給藥。注射后30min處死裸鼠,迅速取出裸鼠腹腔腫瘤小體、腹腔淋巴結(jié)、腹水、血液,高效液相色譜法檢測各組織中的藥物濃度。結(jié)果:TOPLMBs組、OPLMBs組及PTX組腫瘤小體紫杉醇藥量分別為(51.63±6.29μg/m L)、(20.56±5.39μg/m L)和(3.59±0.81μg/m L),腹腔淋巴結(jié)紫杉醇藥量分別為(3.27±0.76μg/m L)、(1.20±0.12μg/m L)和(0.75±0.09μg/m L),血液中紫杉醇藥量分別為(0.56±0.10μg/m L)、(1.19±0.12μg/m L)和(3.11±0.10μg/m L)。腹水中紫杉醇含量分別為(3.25±0.18μg/m L)、(6.51±0.13μg/m L)和(8.96±0.23μg/m L)。腫瘤小體、腹腔淋巴結(jié)中TOPLMBs組紫杉醇藥量均顯著高于OPLMBs組及PTX組(P0.05)。腹水、血液:TOPLMBs組紫杉醇濃度顯著低于OPLMBs組及PTX組(P0.05)。結(jié)論:TOPLMBs能更好的將紫杉醇輸送到腫瘤小體、腹腔淋巴結(jié),在腹水、血液中的藥物濃度小,TOPLMBs有可能提高抗腫瘤效果,減輕全身副作用。第二部分超聲介導(dǎo)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡治療裸鼠卵巢癌腹腔移植瘤的研究目的:探討超聲介導(dǎo)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡對裸鼠卵巢癌腹腔移植瘤生長抑制效應(yīng)及相關(guān)機(jī)制。方法:將56只模型裸鼠隨機(jī)分為7組:(a)對照組,(b)紫杉醇組,(c)紫杉醇+超聲組,(d)攜氧載紫杉醇脂質(zhì)微泡組,(e)攜氧載紫杉醇脂質(zhì)微泡+超聲組,(f)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡組,(g)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡+超聲組,每組8只。最后一次治療后24 h,隨機(jī)處死3只獲取腫瘤小體及腹水細(xì)胞,TUNEL檢測腫瘤細(xì)胞凋亡,CD68免疫組化染色標(biāo)記腫瘤相關(guān)巨噬細(xì)胞;免疫組化檢測血管內(nèi)皮生長因子(VEGF)及腫瘤微血管密度(MVD);流式細(xì)胞儀檢測腹水中FR陽性細(xì)胞的凋亡;余下裸鼠觀察荷瘤裸鼠的生存期。結(jié)果:從(a)組到(g)組的中位生存時(shí)間分別為:31天、37天、36天、31天、41天、32天和52天。與對照組(a)PBS組比較,PTX組、PTX+US組、OPLMBs+US組、TOPLMBs+US組均能顯著延長荷瘤裸鼠生存期(P0.05),其中以TOPLMBs+US組延長生存時(shí)間最多(P0.05)。腫瘤組織中腫瘤細(xì)胞與腫瘤相關(guān)巨噬細(xì)胞凋亡:與PBS組比較,PTX組、PTX+US組、OPLMBs+US組、TOPLMBs+US組細(xì)胞凋亡率顯著增高(P0.05),其中TOPLMBs+US組腫瘤細(xì)胞與腫瘤相關(guān)巨噬細(xì)胞凋亡率最高(P0.05)。腫瘤組織中VEGF表達(dá)及MVD生成情況:與PBS組相比,PTX組、PTX+US組、OPLMBs+US組、TOPLMBs+US組的VEGF及微血管密度明顯降低(P0.05),以TOPLMBs+US組最低(P0.05)。從(a)組到(g)組的腹水中葉酸受體陽性細(xì)胞凋亡率分別為:(5.84±0.28)%,(18.72±3.44)%,(18.46±0.80)%,(7.63±0.90)%,(37.88±8.04)%,(7.34±1.08)%和(86.62±2.38)%,TOPLMBs+US組葉酸受體陽性細(xì)胞凋亡率顯著高于各組(P0.05)。結(jié)論:超聲介導(dǎo)葉酸靶向攜氧載紫杉醇脂質(zhì)微泡能顯著延長荷瘤裸鼠的生存期,主要通過雙靶向殺傷腫瘤細(xì)胞與腫瘤相關(guān)巨噬細(xì)胞、改善腫瘤乏氧微環(huán)境并抑制腫瘤血管生成來實(shí)現(xiàn)。
[Abstract]:The first part: the synthesis of folate targeted oxygen carrying paclitaxel carrying liposome microbubbles in vivo detection and targeting ability of the first control preparation of folic acid targeted paclitaxel load to the oxygen carrying alcohol lipid microbubbles and in vivo targeting ability analysis objective: to synthesize folate targeted oxygen carrying paclitaxel carrying liposome microbubbles, explore the body of TOPLMBs targeting ability methods: nude mice of human ovarian carcinoma SKOV3 transplanted tumor model, observe the growth of animal tumor model, obtaining tumor corpuscle, HE staining, folate receptor was detected by immunohistochemistry in tumor tissues (FR). The expression of the mechanical vibration method were prepared by oxygen carrying paclitaxel carrying liposome microbubbles (OPLMBs) and folate targeted oxygen carrying paclitaxel carrying liposome microbubbles (TOPLMBs), Di was labeled with I fluorescence. The ovarian cancer model of nude mice were randomly divided into OPLMBs group, TOPLMBs group and TOPLMBs+ folic acid group closed a total of 3 groups. The fluorescence labeled TOPLMBs were treated with OPLMBs. After administration, 30 min, 24 h and 48 h were sacrificed and the tumor ascites cells and tumor bodies; folic acid group closed after adding fluorescent labeled folic acid blocking TOPLMBs, 30 min after administration with ascites and tumor cells were obtained by confocal microscopy, fluorescence distribution from tumor bearing mice were randomly divided. Into two groups: TOPLMBs group and OPLMBs group, microbubble was given for the same fluorescent marker, obtain ascites cells, fluorescence uptake analysis of ovarian cancer cells and peritoneal macrophages by flow cytometry. Results: ovarian cancer xenograft in nude mice tumor was 100%, the average time of tumor formation in nude mice was 8.2. 0.84 days, tumor tissue high expression of folate receptor. Fluorescence microscopy sections: 30min after administration, 24h and 48h, the fluorescence distribution of TOPLMBs group was significantly higher than that of group OPLMBs, group TOPLMBs has the strongest expression in 30min fluorescence. 30min after administration of folic acid fluorescence blocking group The expression of weak expression, but no significant difference with OPLMBs group. Flow cytometry: macrophage uptake fluorescence group TOPLMBs, 5 times stronger than that of OPLMBs group (P0.05), human ovarian cancer SKOV3 cell uptake of fluorescence in TOPLMs group is 3 times stronger than that of OPLMBs group (P0.05). Conclusion: human ovarian cancer cell line SKOV3 in abdominal cavity transplantation tumor model can be successfully established, TOPLMBs and abdominal tumor bodies and ascites cells, tumor associated macrophages can be a very good combination, TOPLMBs can enter the tumor tissue in vivo.TOPLMBs has good targeting ability. Section second: folate targeted oxygen carrying paclitaxel lipid microbubbles in nude mice tissue Objective: to investigate the distribution of folate targeted oxygen carrying paclitaxel carrying liposome microbubbles, definite irradiation time after treatment. Methods: the tumor bearing mice were randomly divided into 3 groups: (a) paclitaxel group (PTX), (b) oxygen carrying paclitaxel carrying liposome microbubbles group (OPLMBs) (c) 鍙墮吀闈跺悜鎼烘哀杞界傳鏉夐唶鑴傝川寰場緇，

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