重组人乳铁蛋白抗宫颈癌作用的研究
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摘要
子宫颈癌是妇科最常见的恶性肿瘤之一。在全球范围内,每年约有20多万女性死于宫颈癌。在发展中国家,宫颈癌则属于常见多发的妇科肿瘤,但其病因尚未完全明确。目前治疗宫颈癌大多采用化学合成药物,在治疗过程中易产生毒副作用和耐药性。蛋白类药物以其毒副作用低和不产生耐药性等优点已引起研究者的高度关注。基因治疗已成为癌症治疗领域中的研究热点。
人乳铁蛋白(human lactoferrin,hLTF)是一种隶属于铁结合蛋白家族的糖基化蛋白。它具有多种生理功能,如抑菌;抗病毒感染作用;调节体内铁的平衡;促进铁的传递和吸收;调节机体免疫功能;增强机体抗病能力等作用。近期的研究表明乳铁蛋白还具有抑制人体多种肿瘤细胞的生长、预防及治疗肿瘤的作用,但用药途径多为口服,腺病毒介导的方式国内外尚未见相关报道。
本研究利用复制缺陷型腺病毒介导人乳铁蛋白基因,经过扩增,纯化之后通过体内外试验对Ad-hLTF抗宫颈癌活性及其作用机理进行了研究,获得如下结果:
(1)重组人乳铁蛋白腺病毒(Ad-hLTF)和含绿色荧光蛋白的腺病毒(Ad-GFP)通过腺病毒纯化试剂盒纯化后病毒滴度分别达到1.6×1011pfu/ml和1.45×1011pfu/ml。
(2)外源性乳铁蛋白基因经腺病毒载体成功转入Hela细胞,当感染复数MOI=100时,体外转染效率达到90%以上。
(3)western blot方法检出外源性乳铁蛋白的高表达,与Ad-GFP组和空白对照组相比差异显著有统计学意义(P<0.05)。
(4)Ad-hLTF能显著抑制Hela细胞生长,与分别时间和剂量呈正比。
(5)Ad-hLTF可诱导Hela细胞凋亡及细胞周期Gl期阻滞,与Ad-GFP组和空白对照组比较有显著差异性(P<0.05)。
(6)体内试验结果显示Ad-hLTF组小鼠移植瘤生长速度远小于Ad-GFP组和空白对照组。
(7)免疫组化结果显示,Ad-hLTF组可显著抑制p53表达,上调p19ARF的表达。本试验从体内体外两部分研究Ad-hLTF抗宫颈癌的作用,并从蛋白表达探讨了其诱导肿瘤凋亡的机理,为开发Ad-hLTF作为治疗宫颈癌的手段之一提供了理论依据。
Cervicial cancer is one of the frequent malignant tumors in the department of gynecology. Each year,more than 200 000 women died because of this disease. In developing countries, the cervicial cancer is the first one in gynecological tumors of women; however, we don’t know the etiopathogenisis clearly. The therapy of cervicial cancer mostly uses chemosynthesis drugs at present, but their side effects and multidrug resistance become frequent during treatment. Naturally occurring drugs have been paid more attention by researcher for their low side-effect and no-drug-resistance.So screening naturally occurring drugs of anti-cervicial cancer becomes the investigative hotspot of pharmacology field.
Human lactoferrin (hLTF) is glycosylated protein which belongs to the family of iron-binding protein. It possesses sorts of physiological functions, such as bacteriostasis; viral infection; adjust the ferric balance; promote the ferric transmission and absorption; regulate the immune function; strengthen the disease resistance; Recent studies have shown that lactoferrin has a wide range of inhibition of human tumor cell growth, prevention and treatment of tumors, but for the oral route of administration, adenovirus-mediated approach has yet to see reports at home and abroad.
This study made use of replication-deficient adenovirus-mediated human lactoferrin gene, after amplification, purification ,then through the test in vitro and in vivo to research Ad-hLTF anti-cancer activity and its mechanism and obtain the following results:
(1) Recombinant human lactoferrin adenovirus (Ad-hLTF) and green fluorescent protein adenovirus (Ad-GFP) through the purification of adenovirus purification kits, the virus titer respectively reached 1.6×1011pfu/ml and 1.45×1011pfu/ml.
(2)Exogenous human lactoferrin gene was successfully transfected into Hela cells and the efficiency of adenovirus mediated human lactoferrin gene transfer was more than 90% when the multiplicities of infection(MOI)was 100.
(3)Analysis by western blot on the expression of human lactoferrin in Hela cells showed that the intensity of human lactoferrin in Ad-hLTF group was remarkably strong compared with other control group(P<0.05).
(4)Ad-hLTF significantly suppressed the growth of Hela cells ,which express time-dependent and does-dependent.
(5)Ad-hLTF could induce the apoptosis and arrest cell cycle in G1.Apoptosis rate in Ad-hLTF group was more significant different than Ad-GFP group and control group (P<0.05).
(6)The results in vivo showed that the growth rate of transplanting tumor in Ad-hLTF group is far less than Ad-GFP group and control group.
(7)Immunohistochemistry results showed that Ad-hLTF may significantly inhibits the expression of p53, increases the expression of p19ARF.
The study in vivo and in vitro investigated anti-cervicial cancer effect of Ad-hLTF and discussed the action mechanism from the point of view of apoptosis induced by protein expression. It provided rationale to develop Ad-hLTF as a means of treatment of anti-cervical cancer.
人乳铁蛋白(human lactoferrin,hLTF)是一种隶属于铁结合蛋白家族的糖基化蛋白。它具有多种生理功能,如抑菌;抗病毒感染作用;调节体内铁的平衡;促进铁的传递和吸收;调节机体免疫功能;增强机体抗病能力等作用。近期的研究表明乳铁蛋白还具有抑制人体多种肿瘤细胞的生长、预防及治疗肿瘤的作用,但用药途径多为口服,腺病毒介导的方式国内外尚未见相关报道。
本研究利用复制缺陷型腺病毒介导人乳铁蛋白基因,经过扩增,纯化之后通过体内外试验对Ad-hLTF抗宫颈癌活性及其作用机理进行了研究,获得如下结果:
(1)重组人乳铁蛋白腺病毒(Ad-hLTF)和含绿色荧光蛋白的腺病毒(Ad-GFP)通过腺病毒纯化试剂盒纯化后病毒滴度分别达到1.6×1011pfu/ml和1.45×1011pfu/ml。
(2)外源性乳铁蛋白基因经腺病毒载体成功转入Hela细胞,当感染复数MOI=100时,体外转染效率达到90%以上。
(3)western blot方法检出外源性乳铁蛋白的高表达,与Ad-GFP组和空白对照组相比差异显著有统计学意义(P<0.05)。
(4)Ad-hLTF能显著抑制Hela细胞生长,与分别时间和剂量呈正比。
(5)Ad-hLTF可诱导Hela细胞凋亡及细胞周期Gl期阻滞,与Ad-GFP组和空白对照组比较有显著差异性(P<0.05)。
(6)体内试验结果显示Ad-hLTF组小鼠移植瘤生长速度远小于Ad-GFP组和空白对照组。
(7)免疫组化结果显示,Ad-hLTF组可显著抑制p53表达,上调p19ARF的表达。本试验从体内体外两部分研究Ad-hLTF抗宫颈癌的作用,并从蛋白表达探讨了其诱导肿瘤凋亡的机理,为开发Ad-hLTF作为治疗宫颈癌的手段之一提供了理论依据。
Cervicial cancer is one of the frequent malignant tumors in the department of gynecology. Each year,more than 200 000 women died because of this disease. In developing countries, the cervicial cancer is the first one in gynecological tumors of women; however, we don’t know the etiopathogenisis clearly. The therapy of cervicial cancer mostly uses chemosynthesis drugs at present, but their side effects and multidrug resistance become frequent during treatment. Naturally occurring drugs have been paid more attention by researcher for their low side-effect and no-drug-resistance.So screening naturally occurring drugs of anti-cervicial cancer becomes the investigative hotspot of pharmacology field.
Human lactoferrin (hLTF) is glycosylated protein which belongs to the family of iron-binding protein. It possesses sorts of physiological functions, such as bacteriostasis; viral infection; adjust the ferric balance; promote the ferric transmission and absorption; regulate the immune function; strengthen the disease resistance; Recent studies have shown that lactoferrin has a wide range of inhibition of human tumor cell growth, prevention and treatment of tumors, but for the oral route of administration, adenovirus-mediated approach has yet to see reports at home and abroad.
This study made use of replication-deficient adenovirus-mediated human lactoferrin gene, after amplification, purification ,then through the test in vitro and in vivo to research Ad-hLTF anti-cancer activity and its mechanism and obtain the following results:
(1) Recombinant human lactoferrin adenovirus (Ad-hLTF) and green fluorescent protein adenovirus (Ad-GFP) through the purification of adenovirus purification kits, the virus titer respectively reached 1.6×1011pfu/ml and 1.45×1011pfu/ml.
(2)Exogenous human lactoferrin gene was successfully transfected into Hela cells and the efficiency of adenovirus mediated human lactoferrin gene transfer was more than 90% when the multiplicities of infection(MOI)was 100.
(3)Analysis by western blot on the expression of human lactoferrin in Hela cells showed that the intensity of human lactoferrin in Ad-hLTF group was remarkably strong compared with other control group(P<0.05).
(4)Ad-hLTF significantly suppressed the growth of Hela cells ,which express time-dependent and does-dependent.
(5)Ad-hLTF could induce the apoptosis and arrest cell cycle in G1.Apoptosis rate in Ad-hLTF group was more significant different than Ad-GFP group and control group (P<0.05).
(6)The results in vivo showed that the growth rate of transplanting tumor in Ad-hLTF group is far less than Ad-GFP group and control group.
(7)Immunohistochemistry results showed that Ad-hLTF may significantly inhibits the expression of p53, increases the expression of p19ARF.
The study in vivo and in vitro investigated anti-cervicial cancer effect of Ad-hLTF and discussed the action mechanism from the point of view of apoptosis induced by protein expression. It provided rationale to develop Ad-hLTF as a means of treatment of anti-cervical cancer.
引文
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