本文選題：ES2-AF + 化學(xué)修飾��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：內(nèi)皮抑素(Endostatin,ES)是一個(gè)分子量為20kDa的多肽,最早是從小鼠血管內(nèi)皮瘤中分離出來的,成為首個(gè)被發(fā)現(xiàn)的內(nèi)源性抑制血管新生的細(xì)胞外基質(zhì)片段,同時(shí)也是被研究最多的內(nèi)源性血管新生抑制劑。ES中有一段可高效抑制內(nèi)皮細(xì)胞增殖、遷移的序列ES2(60-70,IVRRADRAAVP),其體內(nèi)抑制新生血管生成的活性約為完整ES序列的3倍。Anti-fltl肽(GNQWFI,AF)是從位置掃描合成肽組合文庫中通過一個(gè)包含純化的重組VEGF及嵌合受體的掃描系統(tǒng)遴選出來的一種VEGFR1選擇性的六肽。AF肽可特異地與VEGFR1結(jié)合,從而抑制VEGFR1與一系列配體,如VEGFA、VEGFB、PIGF等的相互作用。與其他VEGFR1的單克隆抗體或RNA拮抗劑不同,AF肽是一種能夠通過較低生產(chǎn)成本簡易合成的非免疫原性的六肽,這些優(yōu)點(diǎn)決定了其適合在臨床應(yīng)用,但是存在水溶性較差的缺點(diǎn)。透明質(zhì)酸(Hyaluronic acid,HA)是一種從牛眼玻璃體中發(fā)現(xiàn)的、帶負(fù)電荷的、非硫酸化的線性糖胺聚糖,重復(fù)二糖單元為β-N-乙酰氨基葡萄糖和D-葡萄糖醛酸,其生物可降解和高載藥性的特點(diǎn)使其廣泛應(yīng)用于醫(yī)藥領(lǐng)域。HA可通過與細(xì)胞及細(xì)胞間質(zhì)的受體作用調(diào)節(jié)腫瘤細(xì)胞侵襲、腫瘤細(xì)胞的分化、成纖維細(xì)胞中病毒的轉(zhuǎn)化、胚胎組織形成等過程。有四種HA特定受體存在于細(xì)胞膜表面——CD44、RHAMM(Receptor for HA-Mediated Motility)、IVd4 和 LEC,這之中在腫瘤部位表達(dá)較多的是CD44。AF和ES2在活性方面既有相似又有不同,如果將這兩個(gè)肽的序列融合在同一個(gè)肽中,有望獲得更好的生物活性、更好的穩(wěn)定性和更長的半衰期。為此,我們?cè)O(shè)計(jì)了序列為IVRRADRAAVPGGGGGGNQWFI的肽鏈,將其命名為ES2-AF,并利用可以靶向腫瘤部位高表達(dá)的CD44受體的HA對(duì)其進(jìn)行化學(xué)修飾,期待修飾后的多肽溶解性更好、靶向性更強(qiáng)、半衰期更長,從而更好的在體內(nèi)更好的發(fā)揮抗新生血管生成作用。本課題研究內(nèi)容和取得的主要成果如下:1..ES2-AF的合成與表征采用Fmoc固相合成方法合成了一種ES2-linker(GGGG)-AF肽,由高效液相色譜進(jìn)行純化,純度均可達(dá)95%以上,并進(jìn)行了結(jié)構(gòu)的確證。2.HA-ES2-AF的合成與表征由于HA溶于水但不溶于有機(jī)溶劑,為了下一步的偶聯(lián)反應(yīng),首先用四丁基氫氧化銨對(duì)其進(jìn)行離子交換,形成可溶于DMSO的HA-TBA結(jié)合物。HA-TBA分子經(jīng)卡特縮合劑(BOP)活化羧基后,與ES2-AF、DIPEA(N,N-二異丙基乙胺)反應(yīng)得到HA-ES2-AF結(jié)合物。經(jīng)1H NMR核磁結(jié)果分析表明ES2-AF成功被HA修飾,連接率93.75%,平均每HA鏈上連結(jié)60個(gè)ES2-AF肽。3.ES2-AF與HA-ES2-AF的生物活性研究3.1 ES2-AF及HA-ES2-AF的體外抗新生血管生成活性研究(1)采用MTT法評(píng)價(jià)了抗內(nèi)皮細(xì)胞增殖作用,ES2與ES2-AF在體外均具有抑制內(nèi)皮細(xì)胞增殖的作用,并呈現(xiàn)隨濃度增加的趨勢。當(dāng)ES2-AF濃度分別為5μg/mL、50 μg/mL、1000μg/mL、2000μg/mL、500μg/mL 時(shí),其對(duì)內(nèi)皮細(xì)胞增殖的抑制率分別為(11.37%±6.09%)、(13.63%±4.92%)、(17.61%±5.59%)、(16.74%±4.57%)、(27.49%士5.56%)。經(jīng) HA 修飾后的結(jié)合物 HA-ES2-AF 也在體外表現(xiàn)出了一定的抑制內(nèi)皮細(xì)胞增殖的作用,當(dāng)其肽濃度分別為表5 μg/mL、50μg/mL、100μg/mL、200μg/mL、5000μg/mL時(shí),其對(duì)內(nèi)皮細(xì)胞增殖的抑制率分別為(6.67%±4.92%)、(12.01%±8.13%)、(16.96%±5.23%)、(24.37%±8.97%)、(36.40%±7.65%)。(2)采用Transwell小室法測定了抑制內(nèi)皮細(xì)胞轉(zhuǎn)移的作用。以PBS作為空白對(duì)照,當(dāng)藥物肽濃度均為200 μg/mL時(shí),AF、ES2與ES2-AF三組發(fā)生細(xì)胞轉(zhuǎn)移的個(gè)數(shù)分別為(229±11)、(254±4)、(255±13)。AF、ES2 與 ES2-AF 三組藥物對(duì)于內(nèi)皮細(xì)胞轉(zhuǎn)移都有一定的抑制作用,但是差異不大,說明三組實(shí)驗(yàn)藥物對(duì)于腫瘤轉(zhuǎn)移的抑制作用并沒有很大差異。當(dāng)肽濃度均為200μg/mL時(shí),ES2-AF、HAES2-AF混合物、HA-ES2-AF結(jié)合物三組發(fā)生細(xì)胞轉(zhuǎn)移的個(gè)數(shù)分別為(255±13)、(202±24)、(170±7),HA-ES2-AF 表現(xiàn)出了最強(qiáng)的抑制內(nèi)皮細(xì)胞轉(zhuǎn)移的作用,具體機(jī)制仍有待進(jìn)一步研究。(3)利用管腔形成實(shí)驗(yàn)研究了 ES2-AF、HA-ES2-AF等對(duì)內(nèi)皮細(xì)胞EAhy926形成血管的影響。用PBS作為空白對(duì)照,AF與ES2在各濃度時(shí)抑制體外內(nèi)皮細(xì)胞管腔形成的作用相差不大,但ES2-AF對(duì)于內(nèi)皮細(xì)胞管腔的形成的抑制作用明顯高于AF和ES2。在肽濃度均為25 μg/mL、100 μg/mL、200 μg/mL時(shí),AF組的節(jié)點(diǎn)數(shù)分別為(99± 19)、(75± 17)、(63± 12),ES2組的節(jié)點(diǎn)數(shù)分別為(67± 14)、(72±3)、(61±5),HA-ES2-AF 組的節(jié)點(diǎn)數(shù)分別為(32±3)、(17±6)、(19±5)。由此可見,HA-ES2-AF對(duì)內(nèi)皮細(xì)胞管腔形成的抑制作用在所有給藥組中是最強(qiáng)的,說明利用HA對(duì)多肽的化學(xué)修飾提高了其抑制內(nèi)皮細(xì)胞官腔形成的能力。(4)采用ELISA方法根據(jù)抗原抗體結(jié)合原理測定了 AF、ES2、ES2-AF及HA-ES2-AF對(duì)于VEGF及其受體VEGFR1(Flt-1)結(jié)合的抑制能力。ES2與ES2-AF均表現(xiàn)出比AF更強(qiáng)的抑制能力,其中ES2-AF的抑制作用略強(qiáng)于ES2。HA-ES2-AF在 5 μg/mL～200 μg/mL 范圍內(nèi)的相對(duì)值分別為(83.24%±0%)、(55.01%±0.07%)、(51.04%士0.09%)、(41.25%±0.01%)、(37.76%±0.03%),說明 HA-ES2-AF對(duì)VEGF及其受體VEGFR1(Flt-1)結(jié)合的抑制能力呈濃度依賴性提高。較高濃度時(shí)HA-ES2-AF抑制VEGF及其受體VEGFR1(Flt-1)結(jié)合的能力明顯高于ES2-AF肽。3.2 ES2-AF及HA-ES2-AF的體內(nèi)抗新生血管生成活性研究在雞胚絨毛尿囊膜(CAM)實(shí)驗(yàn)中,與生理鹽水組進(jìn)行相比,AF在實(shí)驗(yàn)劑量下對(duì)CAM血管生成沒有明顯抑制作用;ES2-AF組在5 μg/mL、25 μg/mL、50 μg/mL時(shí)新生血管面積百分比分別為(32.03%±0.10%)、(22.84%±0.19%)、(19.26%±0.03%),表現(xiàn)出比ES2更高的體內(nèi)抗血管生成的生物活性并呈現(xiàn)劑量相關(guān)。HA與ES2-AF混合物中HA的加入并沒有對(duì)ES2-AF在動(dòng)物體內(nèi)的抑制CAM血管生成產(chǎn)生較大影響。而HA-ES2-AF組在5 μg/mL、25 μg噸/mL、50 μg/mL時(shí)新生血管面積百分比分別為(9.67%±0.03%)、(10.12%±0.01%)、(13.10%±0.04%),與ES2-AF及混合物組相比抑制CAM血管生成的活性明顯提高。4.HA-ES2-AF的靶向能力研究4.1體外與CD44受體結(jié)合能力研究通過表面等離子共振(SPR)技術(shù)研究了 HA-ES2-AF在體外對(duì)腫瘤細(xì)胞表面受體CD44的結(jié)合能力。以HA為陽性對(duì)照,研究了 HA-ES2-AF、ES2-AF與CD44蛋白之間的相互作用關(guān)系。篩選合適的pH將CD44蛋白錨定于CM5芯片表面,使樣品流過芯片,通過Biacore T200的分析軟件得出以下三種樣品與CD44蛋白結(jié)合的 KD值:HA 為 4.198×10-11 mol/L,HA-ES2-AF 為 4.779×10-10mol/L,ES2-AF為3.011×10-4mol/L,即樣品與CD44結(jié)合能力的排列順序?yàn)?HAHA-ES2-AF》ES2-AF,經(jīng)過HA的修飾HA-ES2-AF結(jié)合物對(duì)細(xì)胞表面受體CD44的結(jié)合能力較修飾前的肽有十分顯著的提升,具有潛在的體內(nèi)腫瘤部位靶向能力。4.2體內(nèi)組織分布研究通過活體成像技術(shù)研究了 HA-ES2-AF在體內(nèi)組織分布情況。將ES2-AF與HA-ES2-AF用FITC標(biāo)記,分別給藥于荷黑色素瘤裸鼠,采用小動(dòng)物活體成像儀進(jìn)行拍照記錄。通過比較發(fā)現(xiàn)HA-ES2-AF-FTIC在裸鼠體內(nèi)分布代謝時(shí)間較長,即HA-ES2-AF-FITC的體內(nèi)血漿半衰期較長,證明了化學(xué)修飾有助于增強(qiáng)多肽的穩(wěn)定性,這與后面藥代動(dòng)力學(xué)實(shí)驗(yàn)的結(jié)果一致;分布于腫瘤部位的藥物增多,表明經(jīng)HA修飾的ES2-AF-FITC在荷瘤裸鼠體內(nèi)對(duì)于腫瘤部位有一定的靶向性,證明了糖基化修飾有助于提高多肽的靶向分布能力,這與SPR實(shí)驗(yàn)的結(jié)果相符合。5.HA-ES2-AF的藥代動(dòng)力學(xué)研究本實(shí)驗(yàn)選用Wistar大鼠作為體內(nèi)藥物代謝動(dòng)力學(xué)的模型,通過比較藥代動(dòng)力學(xué)參數(shù)發(fā)現(xiàn),與ES2-AF相比,HA-ES2-AF在大鼠體內(nèi)的循環(huán)時(shí)間明顯延長,半衰期由2.79 h延長至18.07 h,平均滯留時(shí)間MRT由4.68 h增加至13.18 h,藥時(shí)曲線下面積AUC0-∞也由2887.80 mg/L·h增大至10694.70 mg/L·h,血漿清除率CL也有所下降。較長時(shí)間的維持高血藥水平有助于減少給藥劑量或降低給藥頻率。本研究取得的成果和結(jié)論主要有:(1)成功固相合成了純度在95%以上的ES2-AF肽,其體內(nèi)外抗新生血管生成活性高于ES2或AF。(2)成功完成HA對(duì)ES2-AF的化學(xué)修飾及結(jié)構(gòu)表征,平均每HA鏈上連結(jié)60 個(gè) ES2-AF肽。(3)HA-ES2-AF表現(xiàn)出比ES2-AF和HAES2-AF混合物更強(qiáng)的抑制內(nèi)皮細(xì)胞增殖與遷移的能力、更強(qiáng)的抑制VEGF與受體結(jié)合的能力,以及更強(qiáng)的抑制CAM新生血管生成的能力。(4)利用SPR和活體成像技術(shù)發(fā)現(xiàn),HA-ES2-AF比ES2-AF具有更強(qiáng)的CD44結(jié)合能力,并表現(xiàn)出一定的體內(nèi)腫瘤部位靶向性和更長的體內(nèi)分布代謝時(shí)間。(5)HA-ES2-AF的體內(nèi)半衰期是ES2-AF的6.48倍,有助于減少給藥劑量或降低給藥頻率。
[Abstract]:Endostatin (ES) is a polypeptide of molecular weight of 20kDa. It was first isolated from the mouse endothelioma and became the first endogenous extracellular matrix fragment to inhibit angiogenesis, and it was also the most studied endogenous angiogenesis inhibitor,.ES, which could effectively inhibit the proliferation of endothelial cells. The migration sequence ES2 (60-70, IVRRADRAAVP), the 3 times.Anti-fltl peptide (GNQWFI, AF) that inhibits the angiogenesis activity of the neovascularization in the complete ES sequence (GNQWFI, AF) is a VEGFR1 selective six peptide.AF peptide selected from the position scanning synthetic peptide library through a purified recombinant VEGF and chimeric receptor. Unlike VEGFR1, which inhibits the interaction of VEGFR1 with a series of ligands, such as VEGFA, VEGFB, PIGF, etc., unlike other VEGFR1 monoclonal antibodies or RNA antagonists, AF peptide is a non immunogenic six peptide that can be easily synthesized through lower production costs. These advantages determine that it is suitable for clinical application, but there is water. Hyaluronic acid (HA) is a kind of negative charged, non sulfated linear glycosaminoglycan found in the vitreous body of the bull's eye, and repeated two sugar units are beta -N- acetylglucosamine and D- glucuronic acid. Their biodegradability and high drug resistance are widely used in pharmaceutical field.HA. The receptor action of cell and cell interstitium regulates the invasion of tumor cells, differentiation of tumor cells, transformation of viruses in fibroblasts, and embryonic tissue formation. There are four kinds of HA specific receptors that exist on the surface of the cell membrane - CD44, RHAMM (Receptor for HA-Mediated Motility), IVd4 and LEC, which are more expressed in the tumor site. CD44.AF and ES2 are both similar and different in activity. If the sequences of these two peptides are fused into the same peptide, better bioactivity, better stability and longer half-life are expected. To this end, we designed a peptide sequence of IVRRADRAAVPGGGGGGNQWFI, named ES2-AF, and can be used to target tumor. HA, a highly expressed CD44 receptor, modifies it chemically. It is expected that the modified peptides have better solubility, stronger targeting and longer half-life, and thus better play the role of anti angiogenesis in the body. The contents of this study and the main achievements of this study are as follows: the synthesis and characterization of 1..ES2-AF by solid phase synthesis of Fmoc Methods a kind of ES2-linker (GGGG) -AF peptide was synthesized and purified by high performance liquid chromatography with the purity of up to 95%. The structure confirmed the synthesis and characterization of.2.HA-ES2-AF because HA was dissolved in water but insoluble in organic solvent. For the next step of coupling reaction, four butyl hydrogen hydroxide was first used to exchange it. After activating the carboxyl group.HA-TBA molecule soluble in DMSO HA-TBA binding agent (BOP), the HA-ES2-AF binding was obtained with ES2-AF, DIPEA (N, N- two isopropyl ethylamine). The 1H NMR NMR results showed that ES2-AF successfully was modified and the connection rate was 93.75%. The average biological activity of 60 peptides connected to each chain was found. Study on the antiangiogenesis activity of 3.1 ES2-AF and HA-ES2-AF in vitro (1) the proliferation of endothelial cells was evaluated by MTT. Both ES2 and ES2-AF had the effect of inhibiting the proliferation of endothelial cells in vitro, and showed a tendency to increase with concentration. When ES2-AF concentration was 5 mu g/ mL, 50 u g/mL, 1000 mu g/mL, 2000 mu g/mL, 500 um g/mL, The inhibition rate of endothelial cell proliferation was (11.37% + 6.09%), (13.63% + 4.92%), (17.61% + 5.59%), (16.74% + 4.57%) and (27.49% 5.56%). The binding substance HA-ES2-AF modified by HA also showed a certain inhibitory effect on endothelial cell proliferation in vitro, when its peptide concentration was 5 g/mL, 50 mu g/mL, 100 mu g/mL, 200 mu g/mL, respectively. The inhibition rate of the proliferation of endothelial cells was (6.67% + 4.92%), (12.01% + 8.13%), (16.96% + 5.23%), (24.37% + 8.97%) and (36.40% + 7.65%). (2) the inhibitory effect of inhibition of endothelial cell metastasis was measured by Transwell chamber method. PBS was used as empty white control. When the concentration of drug peptide was 200 g/mL, AF, ES2 and ES2-AF three groups were fine. The number of cell metastases was (229 + 11), (254 + 4), (255 + 13).AF, ES2 and ES2-AF three had a certain inhibitory effect on endothelial cell metastasis, but the difference was not significant. It showed that there was no significant difference in the inhibitory effect of the three groups of experimental drugs on tumor metastasis. When the peptide concentration was 200 g/mL, ES2-AF, HAES2-AF mixture, HA-ES2- The number of cell metastases in the three groups of AF conjugates was (255 + 13), (202 + 24) and (170 + 7), and HA-ES2-AF showed the strongest inhibitory effect on endothelial cell metastasis. The specific mechanism still remained to be further studied. (3) the effect of ES2-AF, HA-ES2-AF and so on on the vascular formation of endothelial cell EAhy926 was studied. PBS was used as an empty space. In white control, the inhibitory effect of AF and ES2 on the formation of endothelium cells in vitro was not very different, but the inhibitory effect of ES2-AF on the formation of endothelium cells was significantly higher than that of AF and ES2. at the concentration of 25 mu g/mL, 100 g/mL and 200 u g/mL, the number of nodes in AF group was (75 + 17), (63 + 12), and the nodes of ES2 group were divided. Not (67 + 14), (72 + 3), (61 + 5), the number of nodes in group HA-ES2-AF was (32 + 3), (17 + 6), (17 + 6), (19 + 5). Thus, the inhibitory effect of HA-ES2-AF on the formation of endothelium cells was the strongest in all drug groups, indicating that the ability of using HA to modify the peptide to inhibit the formation of endothelial cells was improved. (4) the use of ELISA method The inhibition ability of AF, ES2, ES2-AF and HA-ES2-AF to VEGF and its receptor VEGFR1 (Flt-1) binding ability.ES2 and ES2-AF were determined by the antigen antibody binding principle. The inhibitory effect of ES2-AF was stronger than that of ES2.HA-ES2-AF in the range of 5 micron to 200 micron (83.24% + 0%), (55.01% + 0.07%), (51.04% 0.09%), (41.25% + 0.01%), (37.76% + 0.03%), indicating that HA-ES2-AF has a concentration dependence on the inhibition of VEGF and its receptor VEGFR1 (Flt-1) binding. The ability of HA-ES2-AF to inhibit VEGF and its receptor VEGFR1 (Flt-1) binding at higher concentrations is significantly higher than that of ES2-AF peptide.3.2 ES2-AF and HA-ES2-AF in vivo. The activity study in the chick embryo chorioallantoic membrane (CAM) experiment, compared with the saline group, AF did not significantly inhibit the angiogenesis of CAM at the experimental dose, and the percentage of the new blood vessel area in the ES2-AF group was (32.03% + 0.10%), (22.84% + 0.19%) and (19.26% + 0.03%) at 5 UU g/mL, 50 u g/mL, and (19.26% + 0.03%), showing higher than ES2. The biological activity of anti angiogenesis in the body and the addition of HA in the mixture of dose related.HA and ES2-AF did not have a significant effect on the inhibition of ES2-AF in the inhibition of CAM angiogenesis in the animals. The percentage of the HA-ES2-AF group was (9.67% + 0.03%), (10.12% + 0.01%), respectively (10.12% + 0.01%), (13.10% + 0) (13.10% + 0) at 5 mu g/mL, 9.67% tons /mL and 50 micron g/mL. 4%), compared with ES2-AF and mixture group, inhibition of the activity of CAM angiogenesis significantly improved the targeting ability of.4.HA-ES2-AF. 4.1 in vitro and CD44 receptor binding ability study through surface plasmon resonance (SPR) technique to study the binding ability of HA-ES2-AF to the surface receptor CD44 of tumor cells in vitro. With HA as positive control, the study of HA-ES2 The interaction between -AF, ES2-AF and CD44 protein. Select the appropriate pH to anchor the CD44 protein on the surface of the CM5 chip and flow the sample through the chip. Through the analysis software of Biacore T200, the KD values of the following three samples are combined with the CD44 protein: HA is 4.198 x 10-11 mol/L. That is, the sequence of the binding ability of the sample and CD44 is HAHA-ES2-AF>ES2-AF, the binding ability of the HA modified HA-ES2-AF binding to the cell surface receptor CD44 is significantly improved, and the potential target of the tumor location in the body of the body,.4.2 in vivo, is studied by the living imaging technique for the study of HA-ES2-A The tissue distribution of F in the body. ES2-AF and HA-ES2-AF were marked with FITC, and the drug was given to nude mice with melanoma, respectively. The metabolism time of HA-ES2-AF-FTIC in nude mice was longer, that is, the plasma half-life of HA-ES2-AF-FITC in the body was longer, which proved that chemical modification was helpful. To enhance the stability of the polypeptide, this is in accordance with the results of the pharmacokinetic experiment in the rear. The increase of the drug distribution at the tumor site indicates that the HA modified ES2-AF-FITC has a certain target to the tumor site in the nude mice bearing the tumor. It is proved that glycosylation modification helps to improve the target distribution of the polypeptide, which is the result of the SPR experiment. The pharmacokinetics of.5.HA-ES2-AF in this experiment selected Wistar rats as a model of pharmacokinetics in vivo. By comparing pharmacokinetic parameters, it was found that compared with ES2-AF, the cycle time of HA-ES2-AF in rats was obviously prolonged, the half life period was extended from 2.79 h to 18.07 h, and the average retention time MRT increased from 4.68 h to 13. .18 h, the area AUC0- infinity under the drug time curve was also increased from 2887.80 mg/L. H to 10694.70 mg/L. H, and the plasma clearance rate CL decreased. The long time maintenance of high blood drug level was helpful to reduce the dosage or reduce the frequency of drug delivery. The results and conclusions of this study were mainly: (1) a successful solid-phase synthesis of more than 95% of the ES2-AF peptide, The activity of anti angiogenesis in vivo and in vivo was higher than that of ES2 or AF. (2), which successfully completed the chemical modification and structural characterization of HA to ES2-AF. On average, 60 ES2-AF peptides were linked to each HA chain. (3) HA-ES2-AF showed a stronger inhibition of endothelial cell proliferation and migration than ES2-AF and HAES2-AF, and stronger inhibition of the binding ability of VEGF to the receptor. And stronger inhibition of CAM angiogenesis. (4) using SPR and living imaging techniques, HA-ES2-AF has a stronger CD44 binding capacity than ES2-AF, and shows a certain target targeting and longer body metabolism time in the body. (5) the half life of HA-ES2-AF is 6.48 times that of ES2-AF and helps to reduce the dosage of the drug. Dose or decrease the frequency of drug delivery.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96

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