抗胃泌素釋放肽前體論文：(131)Ⅰ標(biāo)記抗胃泌素釋放肽前體單鏈抗體scFv的實(shí)驗(yàn)研究

1、. 抗胃泌素釋放肽前體論文：（131）標(biāo)記抗胃泌素釋放肽前體單鏈抗體scFv的實(shí)驗(yàn)研究【中文摘要】研究:研究（131）I標(biāo)記抗胃泌素釋放肽前體( Pro-gastrin-releasing peptide（31-98）, ProGRP（31-98）)單鏈抗體scFv在正常昆明小鼠及荷小細(xì)胞肺癌裸鼠的體內(nèi)分布規(guī)律,并對荷小細(xì)胞肺癌裸鼠進(jìn)行初步顯像,探討（131）I-anti-ProGRP（31-98） scFv作為特定腫瘤顯像劑的可能性。研究方法:1.利用流式細(xì)胞儀和免疫組化兩種方法分別檢測小細(xì)胞肺癌NCI-H446、宮頸癌Hela、大細(xì)胞肺癌H460和肺腺癌A549細(xì)胞株及其組織中的ProGR

2、P表達(dá)情況。2.采用氯胺-T法標(biāo)記制備（131）I-anti-ProGRP（31-98） scFv;凝膠柱分離法分離純化標(biāo)記產(chǎn)物;紙層析法測定標(biāo)記產(chǎn)物的標(biāo)記率、放化純;將（131）I-anti-ProGRP（31-98）scFv分別置于37水浴箱及與正常人血清混合后在不同時(shí)間點(diǎn)測定放化純以了解其穩(wěn)定性;通過細(xì)胞結(jié)合分析法測定標(biāo)記產(chǎn)物的免疫活性。3.自正常昆明小鼠尾靜脈注射（131）I-anti-ProGRP（31-98） scFv后,于不同時(shí)間點(diǎn)剪斷頸動脈處死小鼠,取血液及各主要臟器組織標(biāo)本,計(jì)算每克組織注射百分劑量率（%ID/g）。利用NCI-H446細(xì)胞建立人小細(xì)胞肺癌荷瘤裸鼠模型,自尾靜

3、脈注射（131）I-anti-ProGRP（31-98） scFv后,于不同時(shí)間點(diǎn)處死裸鼠并取血液、移植瘤及各主要臟器組織標(biāo)本,計(jì)算不同時(shí)間點(diǎn)各臟器組織的%ID/g和瘤體/非瘤體組織放射性計(jì)數(shù)比值（T/NT值）。4.對小細(xì)胞肺癌荷瘤裸鼠模型進(jìn)行（131）I-anti-ProGRP（31-98） scFv初步顯像,觀察移植瘤的大體顯像情況,利用ROI技術(shù)在顯像圖上勾畫移植瘤輪廓并計(jì)算瘤體與對側(cè)相同部位的T/B值。研究結(jié)果:1.流式細(xì)胞儀檢測顯示NCI-H446、Hela、H460及A549細(xì)胞株P(guān)roGRP的表達(dá)率分別為95%、77%、53%和4%。免疫組化檢測結(jié)果顯示,小細(xì)胞肺癌、宮頸癌及大細(xì)

4、胞肺癌組織切片的細(xì)胞胞漿內(nèi)均有二氨基聯(lián)苯胺（DAB）染色陽性顆粒分布,而肺腺癌組織切片中未見明顯染色陽性細(xì)胞。2. （131）I-anti-ProGRP（31-98） scFv標(biāo)記率為93.35±0.67%,標(biāo)記產(chǎn)物純化后即刻放化純?yōu)?8.49±1.21%;在37水浴箱中放置24 h后放化純?yōu)?4.30±0.41%,48 h后測定仍大于90%,與正常人血清充分混合在37水浴箱中放置24 h后放化純?yōu)?3.61±2.19%,48 h測定大于80%;（131）I-anti-ProGRP（31-98） scFv對NCI-H446和A549細(xì)胞株的免疫結(jié)合率分別

5、為85.36%、21.02%。3. （131）I-anti-ProGRP（31-98） scFv在正常昆明小鼠和小細(xì)胞肺癌荷瘤裸鼠體內(nèi)主要通過肝臟、腎臟代謝,血液及各主要臟器組織的清除速度快,腦組織和肌肉組織攝取不明顯,符合單鏈抗體在機(jī)體內(nèi)的一般代謝分布規(guī)律。注射12 h后移植瘤體的%ID/g高于其他臟器及組織,24 h達(dá)到最高為5.38±0.92%。T/NT值隨時(shí)間的延長逐漸升高,至注射后24 h基本達(dá)到最高。4.注射（131）I-anti-ProGRP（31-98） scFv 1 h后,小細(xì)胞肺癌移植瘤部位出現(xiàn)放射性濃聚,隨時(shí)間延長移植瘤部位的放射性濃聚程度逐漸增強(qiáng),至24 h最

6、為清晰。結(jié)論:1. （131）I-anti-ProGRP（31-98） scFv標(biāo)記簡便,標(biāo)記率及放化純高,體內(nèi)外穩(wěn)定性好,主要通過肝腎代謝,血液及各主要臟器廓清速度較快。2. （131）I-anti-ProGRP（31-98） scFv在小細(xì)胞肺癌荷瘤裸鼠體內(nèi)的分布結(jié)果表明,它可在移植瘤組織內(nèi)積聚,瘤體與主要器官的T/NT值隨時(shí)間延長而上升,至24 h到達(dá)最高值。3. （131）I-anti-ProGRP（31-98） scFv有望成為一種新型的小細(xì)胞肺癌放射免疫顯像劑,值得進(jìn)一步深入的研究?！居⑽恼?To evaluate the biodistribution of （131）I l

7、abeled anti-ProGRP（31-98） scFv in healthy Kunming mice, tumor-bearing mice and primarily perform the imaging in nude mice bearing human small cell lung cancer so as to investigate the feasibility of （131）I- anti- ProGRP（31-98） scFv as a new tumor tracer for diagnosing of certain tumors.Methods:1. Th

8、e ProGRP expression in NCI-H446, Hela, H460 and A549 cell lines was detected respectively by flow cytometry and its expression in these tumor tissues was estimated by immunohistochemistry.2. Chloramine-T method was used for （131）I labeling anti-ProGRP（31-98） scFv; （131）I-anti-ProGRP（31-98） scFv was

9、purified by gel column separation method; Labeling efficiency and radiochemical purity were measured by paper chromatography; （131）I-anti-ProGRP（31-98） scFv was incubated with healthy serum at 37, and radiochemical purity was measured at different time to determine the stability of labeled product;

10、Immunocompetence of labeled product was determined with cell conjugation assay. 3. Biodistribution study was performed following an i.v.injection （given via the tailvein） of （131）I-anti-ProGRP（31-98） scFv. Healthy Kunming mice were sacrificed by cutting off carotid artery at designated time points i

11、n group, and the blood and major organs were removed, weighed, and counted in a gamma scintillation counter to determine the %ID/g （percentage of injected dose related to the organ weight） for each radioactive substance. In the same way, major organs and transplanted tumor tissues of nude mice beari

12、ng human small cell lung cancer were sampled for calculating %ID/g and tumor/non-tumor （T/NT） ratios.4. After injection of （131）I-anti-ProGRP（31-98） scFv, continuous images of the nude mice bearing SCLC were initially carried out at different time, and then tumor/base（T/B）ratios were calculated.Resu

13、lts:1. The expression rates of ProGRP in NCI-H446, Hela, H460, A549 cell lines were 95%, 77%, 53% and 4% respectively. The results of immunohistochemistry showed that positive granules （DAB staining） were distributed in intracytoplasm of small cell lung cancer, cervical cancer and large cell lung ca

14、ncer, no obvious staining cells were found in lung adenocarcinoma.2. The labeling rate of （131）I-anti-ProGRP（31-98） scFv was 93.35±0.67%, and the radiochemical purity was 98.49±1.21%. After incubation at 37C for 24 h, the radiochemical purity was 94.30±0.41%, and still maintained abov

15、e 90% at 48 h. Meanwhile, after incubation with healthy human serum for 24 h, the radiochemical purity was 83.61±2.19%, and still maintained above 80% at 48 h. The immunobinding rates of NCI-H446 cell and A549 cell were 85.36% and 21.02%.3. The vivo distribution of （131）I-anti-ProGRP（31-98） scF

16、v in the normal Kunming mice and nude mice bearing human small cell lung cancer showed that the metabolism of （131）I-anti-ProGRP（31-98） scFv mainly depended on liver and kidney with rapid elimination in blood and other main organs, the brain and muscle did not uptake much, and it was consistent with

17、 general scFv. The %ID/g of tumor was obviously higher than that of other organs at 12 h and arrived at top 5.38±0.92% at 24 h post-injection. The T/NT ratio increased gradually and arrived at top at 24 h.4. The tumor of nude mice bearing SCLC was visualized at 1 h after injection of （131）I-ant

18、i-ProGRP（31-98） scFv, The radioactivity in tumor site accumulated gradually and the tumor image became the clearest at 24 h post-injection.Conclusions:1. Labeling of （131）I-anti-ProGRP（31-98） scFv is easy, it not only has high labeling efficiency and radiochemical purity, but also has good stability

19、 in vitro and in vivo. （131）I-anti-ProGRP（31-98） scFv was metabolized mainly in kidney and liver, the blood and other major organs radioactivity of （131）I-anti-ProGRP（31-98） scFv decreased rapidly. 2. The biodistribution in SCLC tumor-bearing mice showed that （131）I-anti-ProGRP（31-98） scFv can be accumulated in tumor, the ratios of T/NT increased as time prolongs and arrived at top at 24 h.3. （131）I-anti-ProGRP（31-98） scFv has the potential to become radioimmunoimaging agent of SCLC. It deserves further study.【關(guān)鍵詞】抗胃泌素釋放肽前體 單鏈抗體 (131)I-anti-ProGRP（31-98） scFv 小細(xì)胞肺癌 放射免疫顯像【備注】索購全文在線