乳豬肝膠原蛋白水解物注射液的研制及對(duì)荷瘤小鼠腫瘤生長(zhǎng)的影響

1、乳豬肝膠原蛋白水解物注射液的研制及對(duì)荷瘤小鼠腫瘤生長(zhǎng)的影響         09-02-01 16:21:00     編輯：studa20                  作者：鄒曉軍 廖漢雄 王耐勤 楊聯(lián)萍，劉彤，鄒清雁【摘要】  目的與方法 摸索乳豬肝膠原蛋白水解物注射液（HCH）的制備方法

2、,同時(shí)對(duì)其進(jìn)行質(zhì)量檢驗(yàn)和初步穩(wěn)定性試驗(yàn)；并觀(guān)察其對(duì)荷瘤昆明小鼠HepA肝瘤和S180纖維肉瘤及小鼠體內(nèi)肝癌H22移植瘤的影響。結(jié)果 HCH生產(chǎn)工藝路線(xiàn)合理，質(zhì)量達(dá)到擬定標(biāo)準(zhǔn)要求。HCH能明顯抑制HepA肝瘤生長(zhǎng)，25、50、100、200 mg·kg-1·d-1的抑制率分別為43.26、55.06、74.71、40.47；對(duì)荷瘤小鼠S180纖維肉瘤25、50、100 mg·kg-1·d-1的抑制率分別為53.87、76.53、58.20，而阿霉素（2 mg·kg-1·d-1 ）陽(yáng)性對(duì)照組的抑制率為62.41；對(duì)小鼠體內(nèi)肝癌H22移植瘤

3、生長(zhǎng)的抑制呈劑量相關(guān)性，50、100、200 mg·kg-1·d-1的抑制率分別為48.1、60.0、74.8，而卡鉑（20 mg·kg-1·d-1 ）陽(yáng)性對(duì)照組的抑制率為63.7。結(jié)論 此法可制備合格的HCH，且適合于大規(guī)模生產(chǎn)；HCH能明顯地抑制荷瘤小鼠腫瘤的生長(zhǎng)。 【關(guān)鍵詞】  膠原；腫瘤；生長(zhǎng)抑制；小鼠Abstract:Objective To investigate the methods to prepare the hepatic collagenous hydrolysates(HCH) for injection, the r

4、udiment stability test, and the quality control study of the HCH, and to observe its effect on the tumor growth in the tumor bearing mice. Methods The dissolvability collagen were extracted from the suckling pig liver with the biochemical approach, the tumor inhibiting polypeptides for injection wer

5、e prepared with the pepsin hydrolysis. The quality criteria of the product were studied. The effects of TIP on the Hep A hepatoma, H22 hepatoma and S180 fibrosarcoma were investigated.Results The products quality reached the quality criteria showed that the production procedure of TIP was suitable.

6、The results showed that TIP could apparently inhibit the growth of Hep A hepatoma in the tumor-bearing mice with the inhibitory rates of 43.26, 55.06, 74.71, 40.47 at the doses of 25, 50, 100, 200 mg·kg-1·d-1 TIP; and inhibit the growth of S180 fibrosarcoma with the inhibitory rates of 53.

7、87, 76.53, 58.20 at the respective doses of 50, 100, and 200 mg·kg-1·d-1 TIP, the relative inhibitory rate of the positive adriamycin（2 mg·kg-1·d-1）control was 62.41%; TIP could apparently inhibit the growth of H22 hepatoma transplanted in the mice at a dose-dependant way with th

8、e inhibitory rates of 48.1, 60.0, 74.8 at the respective doses of 50, 100, and 200 mg·kg-1·d-1 HCH, and the relative inhibitory rate of the positive adriamycin（20 mg·kg-1·d-1） control was 63.7%.Conclusion HCH prepared with the biochemical approach was qualified and suitable for l

9、argescale prodiction. HCH could apparently inhibit the growth of tumor transplanted in the mice.Key words:collagen; tumor; growth inhibition; miceOReilly等1從小鼠血管內(nèi)皮瘤（EOMA）的培養(yǎng)上清中發(fā)現(xiàn)了一種能特異性地抑制毛細(xì)血管內(nèi)皮細(xì)胞生長(zhǎng)的因子，其分子量為20 kD，命名為內(nèi)皮抑素（endostatin, ES）。ES是膠原的N端產(chǎn)物。膠原為血管內(nèi)皮基底膜的組成成份，主要分布于肝、肺、腎等組織，其中肝實(shí)質(zhì)細(xì)胞、膽管上皮細(xì)胞和新生血管內(nèi)皮細(xì)胞

10、能合成和分泌膠原。根據(jù)以上研究結(jié)論，我們?cè)O(shè)計(jì)了從肝組織中提取可溶性膠原，經(jīng)水解后制備腫瘤生長(zhǎng)抑制物的研究思路2。現(xiàn)將實(shí)驗(yàn)結(jié)果總結(jié)如下。1材料和方法11HCH的制備    取新鮮的乳豬肝去掉血管及筋膜,用注射用水清洗后勻漿，水浴加熱至80 離心；沉渣加10稀鹽酸調(diào)pH值為45，再加胃蛋白酶后于4 保存20 h；用0.1 mol·L-1氫氧化鈉調(diào)pH值到7.0，離心，取上清液；再用10 kD的超濾膜超濾，混合，除菌，分裝，檢驗(yàn)合格后，即制成HCH。12分子量范圍測(cè)定       采用SDS-PAGE

11、D電泳法，具體方法與步驟參見(jiàn)文獻(xiàn)313初步穩(wěn)定性試驗(yàn)    對(duì)3批產(chǎn)品的初步穩(wěn)定性試驗(yàn)主要從溫度、光照、加速試驗(yàn)等3個(gè)方面進(jìn)行考察。進(jìn)行4 留樣觀(guān)察、常溫試驗(yàn)、高溫試驗(yàn)；考察項(xiàng)目有：外觀(guān)、澄清度、蛋白定性、pH值、多肽含量、活性、熱原試驗(yàn)、安全試驗(yàn)、無(wú)菌試驗(yàn)等，其檢測(cè)按擬定的質(zhì)量標(biāo)準(zhǔn)進(jìn)行。14HCH中RNA含量測(cè)定采用定糖法測(cè)定4。15HCH對(duì)荷瘤昆明小鼠HepA肝瘤和S180纖維肉瘤的抑瘤活性測(cè)定151HepA小鼠肝瘤細(xì)胞株（腹水型）和S180小鼠肝瘤細(xì)胞株（腹水型），引自中山大學(xué)實(shí)驗(yàn)動(dòng)物中心。昆明小鼠：雄性，動(dòng)物生產(chǎn)許可證編號(hào)：SCXK（粵）2003-0001

12、，購(gòu)自廣州中醫(yī)藥大學(xué)實(shí)驗(yàn)動(dòng)物中心。152腫瘤細(xì)胞小鼠腹腔接種用含10小牛血清的DME/F12培養(yǎng)液培養(yǎng)，收集細(xì)胞，用無(wú)菌Hanks液將細(xì)胞稀釋成1×1092×109個(gè)/L，進(jìn)行昆明小鼠（體質(zhì)量2630 g）腹腔接種，每鼠0.3 mL，710 d后進(jìn)行試驗(yàn)。153制成小鼠荷瘤模型將有腹腔接種腫瘤細(xì)胞710 d的小鼠斷頸處死，抽取腹水制備1×1092×109個(gè)/L濃度的細(xì)胞懸液，活細(xì)胞數(shù)應(yīng)大于85。取該懸液0.1 mL種植于每只小鼠（體質(zhì)量1824 g）腰部正中線(xiàn)皮下。隨機(jī)分組。154種植后第7天開(kāi)始給藥。荷HepA肝瘤小鼠，陰性對(duì)照組每只小鼠腹腔注射生理鹽

13、水0.5 mL·d-1，陽(yáng)性對(duì)照組腹腔注射阿霉素2 mg·kg-1·d-1，治療組分4個(gè)劑量組分別腹腔注射HCH 25、50、100、200 mg·kg-1·d-1。末次給藥后次日，拉頸處死動(dòng)物，稱(chēng)體質(zhì)量，刨出實(shí)體瘤，稱(chēng)瘤重，計(jì)算抑瘤率。16HCH對(duì)ICR種小鼠體內(nèi)肝癌H22移植瘤的抑瘤活性測(cè)定161ICR種小鼠，1822 g，雌雄各半，購(gòu)自北京維通利華實(shí)驗(yàn)動(dòng)物有限公司，動(dòng)物生產(chǎn)許可證編號(hào)：2002（京）0003.SCXK。飼養(yǎng)于北京大學(xué)人民醫(yī)院SPF級(jí)動(dòng)物實(shí)驗(yàn)室內(nèi)，許可證號(hào)：SYXK11000001。小鼠每籠10只，自由進(jìn)食飲水。小鼠肝癌H22為昆明種小鼠傳代保種的腹水型瘤株，實(shí)驗(yàn)時(shí)抽取第7天的癌性腹水，經(jīng)生理鹽水按13稀釋成瘤細(xì)胞懸液。162取ICR小鼠50只，于右腋部皮下接種H22細(xì)胞懸液，每只0.2 mL。接種后將小鼠隨機(jī)均分為5組，每組10只。次日，開(kāi)始給藥，實(shí)驗(yàn)組共3組，劑量分別為