CN107236032A - 一种从脐带组织中提取复合细胞因子的方法 - Google Patents
一种从脐带组织中提取复合细胞因子的方法 Download PDFInfo
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Abstract
本发明提供一种从脐带组织中提取复合细胞因子的方法,所述方法包括以下步骤:1)脐带组织的处理、2)复合细胞因子的富集、3)复合蛋白因子的分离、4)将步骤3)得到的上清液加压过滤,并进一步使用3KD浓缩脱盐,既得。本发明还提供了一种从脐带组织中提取的复合细胞因子。本发明直接从脐带组织中获得复合细胞因子,这些复合细胞因子均为人体成分,可以直接被皮肤吸收,避免过敏反应,无副作用。本发明的方法不涉及高温及化学灭菌的方式,仅仅利用过滤法去除微生物污染,既避免了产品出现污染变质问题,又保证生物活性成分不被高温、辐射或化学杀菌剂改变结果,保证了产品中细胞因子的活性,使产品效果稳定。
Description
技术领域
本发明属于生物技术领域,涉及一种从脐带组织中提取复合细胞因子的方法,本发明还涉及本发明提取的复合细胞因子的保存方法及其用途。
背景技术
细胞因子是一类通过与特异的、高亲和的细胞膜受体结合,调节细胞生长并参与细胞功能等多效应的多肽类物质,对不同种类细胞具有一定的专一性,由多种维生素、氨基酸、多醣类成分、复杂蛋白质体、醣蛋白等组成。细胞因子由正常细胞分泌,分泌方式主要包括自分泌(autocrine)和旁分泌(paracrine),具有多效性、重叠性、协同性、拮抗性和双重性等特点,该特点是其具有非常重要实验研究价值,有助于阐明分子水平的免疫调节机理,有助于疾病的预防、诊断和治疗,特别是利用基因工程技术生产的重组细胞因子已用于治疗肿瘤、感染、炎症、造血功能障碍等。目前实验研究结果表明其可应用于细胞培养和3D打印的生物材料,具有抗衰老和免疫调节等作用,未来具有非常广阔的应用前景。
间充质干细胞(Mesenchymal stem cells)是一类具有多向分化潜能的成体干细胞,源于发育早期的中胚层,主要存在于结缔组织和器官间质中,可以从骨髓、外周血、脂肪及皮肤等多种组织中获得。间充质干细胞属于非终末分化细胞,它既有间质细胞的特征,又有内皮细胞及上皮细胞的特征;作为一类多能干细胞,它在体外特定的诱导条件下,可以向骨、软骨、肌肉、肌腱、肝、脂肪、神经、内皮及胰岛样细胞等方向增殖分化。目前已知对心肌梗死、糖尿病、骨质疏松、骨坏死、狼疮性肾炎、肝硬化、肝衰竭、多发性肝硬化症、系统性红斑狼疮、帕金森病、脊髓损伤等都有一定治疗效果。
近来有学者从分娩后的废弃物-脐带中分离培养出间充质干细胞,并进行了形态学、分化功能和表面标志物等生物学表型鉴定,确定了这种新型间充质干细胞的开发潜质。这种新型间充质干细胞与骨髓间充质干细胞相比具有明显的优势:(1)来源充足,取材方便,无伦理学争议;(2)含量丰富,可在体外进行分离、培养,且生物学性状稳定,多次传代扩增仍能保持旺盛功能;(3)免疫原性极低;(4)分泌能力强,在增殖生长过程中会产生大量细胞因子等。
目前,从脐带中获得的间充质干细胞除应用于临床研究与治疗外,干细胞分泌产生的各种多肽类细胞因子也越来越被关注。在干细胞的培养液中含有多种干细胞分泌的细胞因子,如干细胞因子(SCF)、肝细胞生长因子(HGF)、神经生长因子(NGF)、基质细胞源性生长因子(SDF)、血管内皮细胞生长因子(VEGF)、血小板源性生长因子(PDGF)、碱性成纤维细胞生长因子(bFGF)、胰岛素样生长因(IGF)、表皮生长因子(EGF)、白细胞介素(IL)、巨核细胞集落刺激因子(M-CSF)、肿瘤坏死因子(TNF)、干扰素(IFN)等,通过多种因子的协同作用可有效调控机体细胞信号传导、活化人体干细胞,进而生理性修复或替代机体损伤、病变及衰老的细胞等。
美国科学家LEVI博士就发现干细胞分泌的细胞因子具有除皱作用,将多种细胞因子组成的生物活性复合蛋白作用于皮肤真皮层的成纤维干细胞,使处于休眠、损伤的成纤维干细胞被激活和修复,并分化成大量成熟的成纤维细胞用于皮肤更新。
然而,当前对细胞因子的开发,首先需要大量扩增间充质干细胞,而原代细胞的活性和数量无疑是最关键的,但目前获取原代细胞的组织块直接贴壁法和酶消化法均略显不足。在酶消化法中,使用多种消化酶(CN102127522B、CN101575590B)进行长时间组合消化获取单细胞悬液(胰酶和胶原酶Ⅱ、Ⅳ),消化方式强烈,易对细胞膜造成破坏,致使细胞变形,增殖能力下降,传代次数减少;且多种酶组合消化的消化时间不易把握,最佳配比有待实验确认。此外多种酶的使用也增加了外源物质引入的风险。而单纯使用组织块培养法则培养时间过长,至少需要20天原代细胞才可以进行传代。原代培养时间的延长将会使细胞长时间暴露于体外环境,增加了细胞的不稳定性,使细胞易发生老化,增殖能力减弱,基因突变几率大幅增加。
因此,当前对更为有效的细胞因子的生产方法存在需求。
发明内容
基于此,本发明的目的是针对现有技术的不足,提供一种从脐带组织中提取复合细胞因子的方法。本发明的方法操作简单、可重复性高、成本低、提取效率高、无化学试剂残留,为复合细胞因子进一步地应用于细胞培养、组织修复、医疗美容、生物3D打印领域提供了基础。
一方面,本发明提供了一种从脐带组织中提取复合细胞因子的方法,所述方法包括以下步骤:
1)脐带组织的处理:
将脐带组织消毒并处理为0.1~0.3cm3的组织块;
2)复合细胞因子的富集:
在步骤1)得到的组织块中加入2~5倍体积生理盐水,离心取沉淀,然后加入1~10倍体积的生理盐水,混合均匀,并置于培养箱中孵化1~6天;
3)复合蛋白因子的分离:
收集步骤2)孵化后的组织块和上清液,离心取上清液;
4)将步骤3)得到的上清液加压依次通过120μm、70μm、50μm、25μm的滤器,并进一步使用2~4KD的过滤膜浓缩脱盐,即得。
优选地,在步骤1)中,所述脐带组织保存在4℃;
优选地,在步骤1)中,所述消毒包括以下的步骤:
将脐带组织使用碘伏浸泡1~10分钟,优选地1~3分钟,更优选地1分钟后,使用体积比为75%的酒精浸泡脱碘,然后使用生理盐水冲洗1~5次,优选地3次。
优选地,在步骤2)中,所述离心的条件为:在1000~1400r/min下,离心4~7min;更优选地,所述离心的条件为:在1000r/min下,离心5min;
优选地,在步骤2)中,离心取沉淀后,加入2~5倍体积,优选地3倍体积的生理盐水混合均匀;优选地,在步骤2)中,混合均匀后,首先将混匀的组织块转移至培养瓶中,然后置于培养箱中孵化;
优选地,在步骤2)中,所述培养箱的孵化条件为:
37℃、CO2的浓度为3%;
优选地,在步骤2)中,在培养箱中孵化2~3天;
优选地,在步骤3)中,所述离心的条件为:在1000~1400r/min下,离心4~7min;更优选地,所述离心的条件为:在1400r/min下,离心5min;
优选地,在步骤4)中,将步骤3)得到的上清液加压依次通过120μm、70μm、50μm、25μm的滤器,过滤条件为进0.3~0.6mpa,温度为5±3℃;
优选地,在步骤4)中,收集通过过滤器的上清液,使用截留分子量3KD过滤膜进行浓缩脱盐。
优选地,收集步骤3)离心后的脐带组织,重复使用1~3次。
另一方面,本发明提供了一种保存从脐带组织中提取的复合细胞因子的方法,将所述复合细胞因子分装于西林瓶中,每瓶5ml。在3h~6h内,冷冻到﹣50℃,抽真空干燥后,使温度上升到5±3℃,可长期保存备用。
使用时,将所述冻干的复合细胞因子加入2-4倍体积的灭菌用水溶解。例如将所述冻干的复合细胞因子每5ml加入10~20ml灭菌用水溶解。
再一方面,本发明提供了本发明的从脐带组织中提取的复合细胞因子;
优选地,所述复合细胞因子包括干细胞因子(SCF)、基质细胞衍生因子-1(SDF-1)、血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、胰岛素样生长因子(IGF)、白介素-6(IL-6)、白介素-8(IL-8)、单核细胞趋化蛋白-1(MCP-1)、金属蛋白酶移植因子(TIMP-1)。
再另一方面,本发明提供了使用本发明的方法提取的复合细胞因子或者本发明的复合细胞因子在制备用于细胞培养、组织修复、医疗美容、生物3D打印的材料或组合物中的用途。
本发明还提供了一种用于细胞培养、组织修复、医疗美容或生物3D打印的材料或组合物,其包含使用本发明的方法提取的复合细胞因子或者本发明的复合细胞因子。
本发明利用生物技术美容,将复合细胞因子用于外用美容,通过补充促进表皮细胞代谢和增殖的生物活性因子,从根本上改善皮肤的生理状态,促进表皮组织的修复和代谢,促进细胞外某些生物大分子(如透明质酸、糖蛋白等)的合成;可使皮肤异常细嫩年轻;可淡化色斑,平复皱纹;可减轻日晒对皮肤的损伤;还具有显著抗衰老作用,故嫩肤美容效果显著,且安全可靠,无副作用。复合细胞因子相互作用,可显著促进人上皮组织的生成和增殖,美容效果显著增强。
与现有技术相比,本发明具有以下的优点和效果:
1.本发明直接从脐带组织中获得复合细胞因子,这些复合细胞因子均为人体成分,可以直接被皮肤吸收,避免过敏反应,无副作用。
2.本发明的每种细胞因子都为人体的内源细胞因子,具有促进皮肤新陈代谢的功能。
3.本发明人发现,使用本发明的方法得到的细胞复合因子,特别适合用于进行表皮组织的修复和代谢,因此在嫩肤美容,除皱抗斑中具有出乎意料的效果(详见本申请实施例3)
4.本发明的方法不涉及高温及化学灭菌的方式,仅仅利用过滤法去除微生物污染,既避免了产品出现污染变质问题,又保证生物活性成分不被高温、辐射或化学杀菌剂改变结果,保证了产品中细胞因子的活性,使产品效果稳定。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
实施例1提取复合细胞因子
所述脐带组织购自三甲医院妇产科,
1)脐带组织的处理:选取无传染病源的脐带组织,4℃保存碘伏浸泡1分钟后,75%酒精浸泡脱碘,0.9%生理盐水冲洗3次,将脐带组织利用机械力剪切成0.1~0.3㏄大小的组织块。
2)复合蛋白因子的富集:收集步骤1)处理得到的组织块,加入适量生理盐水1000r/min离心5min一次,然后加入0.9%生理盐水到收集的组织块中(体积比1∶3=组织块∶0.9%生理盐水)混匀,将混匀的组织块,取适量转移至培养瓶中,置于37℃、3%CO2培养箱中孵化。
3)复合蛋白因子的分离:收集步骤2)的孵化2~3天后的组织块和上清液,1400r/min离心5min一次,收集上清液备用
4)复合蛋白因子的收集保存:将步骤3)得到的上清液加压分别通过120μm、70μm、50μm、25μm的滤器,过滤条件为进0.3~0.6mpa,温度为5±3℃,收集过滤器后的上清液截留分子量3KD过滤膜进行浓缩脱盐,既得液体。
保存时,首先将所述复合细胞因子在3h~6h内冷冻到﹣50℃,抽真空干燥使温度上升到5±3℃,然后长期5±3℃保存备用。
实施例2本发明的复合细胞因子成分
本发明获得的为多种复合细胞因子的复合物,根据生物活性蛋白的性质及主要功能选取5个代表性指标检测生物活性蛋白含量的变化。
应用酶联免疫分析技术,分别检测细胞培养上清中干细胞因子(SCF)、基质细胞衍生因子-1(SDF-1)、血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、胰岛素样生长因子(IGF)、白介素-6(IL-6)、白介素-8(IL-8)、单核细胞趋化蛋白-1(MCP-1)、金属蛋白酶移植因子(TIMP-1)的水平,具体步骤如下。
(1)标准品的稀释与加样:在酶标包被板上设置标准品10个孔,于第一、第二孔中分别加入标准品100μl,然后在第一、第二孔中分别加入标准品稀释液50μl,混匀,混匀后从第一、第二孔中分别取100μl,然后分别加到第三、第四孔,然后在第三、第四孔中分别加入标准品稀释液50μl混匀,然后在第三、第四孔中先分别各取50μl弃掉,然后再各取50μl分别加到第五、第六孔中,并在第五、第六孔中加入标准品稀释液50μl,混匀后从第五、第六孔中各取50μl分别加到第七、第八孔中,再在第七、第八孔中分别加入标准品稀释液50μl,混匀后从第七、第八孔中各取50μl分别加到第九、第十孔,然后再在第九、第十孔中分别加入标准品稀释液50μl,混匀,最后从第九、第十孔各取50μl弃掉。(稀释后各孔加样量都为50μl,浓度分别为180ug/L,120ug/L,60ug/L,30ug/L,15ug/L)。
(2)加样:分别设待测样品孔及空白孔对照(空白对照孔不加样品及酶标试剂,其余各步操作相同)。首先在酶标包被板上待测样品孔中分别加入样品稀释液40μl,然后再加入待测样品10μl(样品最终的稀释度为5倍),轻轻晃动混匀。
(3)温育:用封板膜密封板,并置于37℃恒温箱中,温浴30分钟。
(4)配液:将30倍浓缩的洗涤液用蒸馏水稀释30倍后备用。
(5)洗涤:揭掉封板膜,弃去板中的液体,甩干,然后在每孔中加满洗涤液,静置30秒后弃去孔中的液体,并如此重复5次,然后甩干。
(6)加酶:除空白孔外,其余每孔均加入酶标试剂50μl。
(7)温育:用封板膜密封板后,置于37℃恒温箱,温育30分钟。
(8)洗涤:小心揭开封板膜,弃去孔中的液体,甩干,并在每孔中加满洗涤液,静置30秒后弃去液体,如此重复5次,然后甩干。
(9)显色:首先将每孔中分别加入显色剂A 50μl,然后再加入显色剂B50μl,轻轻震荡混匀,然后置于37℃恒温箱中,避光显色15分钟。
(10)终止:显色完成后,将每孔中各加入终止液50μl,终止反应(此时蓝色立即转为黄色)。
(11)测定:终止反应后立即将酶标包被板,放入酶标仪中。以空白孔调零,450nm波长依序测量各孔的吸光度(OD值)。测定均在加终止液后15分钟内完成。
(12)计算:首先以标准物的浓度为横坐标,OD值为纵坐标,在坐标纸上绘出标准曲线。然后再根据样品的OD值,由标准曲线查出其相应的浓度,再乘以稀释倍数,即为样品的实际浓度,或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品的浓度,再乘以稀释的倍数,即为样品的实际浓度。
数据均以表示
实施例3本发明的细胞因子的功能
实验组:选取冻干的复合细胞因子加入10~20ml灭菌用水溶解,浸透空白压缩面膜。
对照组:选取10~20ml灭菌用水,浸透空白压缩面膜。
实验对象:实验志愿者来广州市周边地区,面部皮肤由于痤疮、皮肤炎症、疤痕或衰老而产生色斑及皱纹。实验剔除妊娠、拟妊娠或哺乳期女性,剔除其他皮肤病患者,如日光性皮炎、面部银屑病、脂溢性皮炎及荨麻疹。志愿者在临床观察期禁止食用辛辣、刺激的食品,戒烟、戒酒。志愿者从临床前2周开始,不使用激素类药物和抗过敏药物,不使用辅助护肤品。志愿者自愿参加实验,签订知情同意书。
实验方法:(1)嫩肤、去皱、祛斑试验
选取25~55岁志愿者120人,分成两组,男女对半,每组60人。每组分别使用上述实验组和空白对照组,连续使用一个月,然后评价其效果。
(2)安全性试验
在实验方法(1)试验中,同时观察对皮肤的致敏性、刺激性和其他不良反应。
实验结果:嫩肤、去皱、祛斑试验和安全性试验
尽管在此公开了本发明的各个方面和实施例,但其他方面和实施例对于本领域技术人员而言也是显而易见的。在此公开的各个方面和实施例仅用于说明目的,而非限制目的。本发明的保护范围和主旨仅通过后附的权利要求书来确定。
Claims (11)
1.一种从脐带组织中提取复合细胞因子的方法,所述方法包括以下步骤:
1)脐带组织的处理:
将脐带组织消毒并处理为0.1~0.3cm3的组织块;
2)复合细胞因子的富集:
在步骤1)得到的组织块中加入2~5倍体积生理盐水,离心取沉淀,然后加入1~10倍体积的生理盐水,混合均匀,并置于培养箱中孵化1~6天;
3)复合蛋白因子的分离:
收集步骤2)孵化后的组织块和上清液,离心取上清液;
4)将步骤3)得到的上清液加压依次通过120μm、70μm、50μm、25μm的滤器,并进一步使用2~4KD的过滤膜浓缩脱盐,收集过滤液,即得。
2.根据权利要求1所述的方法,其特征在于,在步骤1)中,所述脐带组织保存在4℃;
优选地,在步骤1)中,所述消毒包括以下的步骤:
将脐带组织使用碘伏浸泡1~10分钟,优选地1~3分钟,更优选地1分钟后,使用体积比为75%的酒精浸泡脱碘,然后使用生理盐水冲洗1~5次,优选地3次。
3.根据权利要求1或2所述的方法,其特征在于,在步骤2)中,所述离心的条件为:在1000~1400r/min下,离心4~7min;更优选地,所述离心的条件为:在1000r/min下,离心5min;
优选地,在步骤2)中,离心取沉淀后,加入2~5倍体积,优选地3倍体积的生理盐水混合均匀;优选地,在步骤2)中,混合均匀后,首先将混匀的组织块转移至培养瓶中,然后置于培养箱中孵化;
优选地,在步骤2)中,所述培养箱的孵化条件为:
37℃、CO2的浓度为3%;
优选地,在步骤2)中,在培养箱中孵化2~3天。
4.根据权利要求1-3中任一项所述的方法,其特征在于,在步骤3)中,所述离心的条件为:在1000~1400r/min下,离心4~7min;优选地,所述离心的条件为:在1400r/min下,离心5min。
5.根据权利要求1-4中任一项所述的方法,其特征在于,在步骤4)中,将步骤3)得到的上清液加压依次通过120μm、70μm、50μm、25μm的滤器,过滤条件为进0.3~0.6mpa,温度为5±3℃;
优选地,在步骤4)中,收集通过过滤器的上清液,使用截留分子量3KD过滤膜进行浓缩脱盐。
6.根据权利要求1-5中任一项所述的方法,其特征在于,所述方法还包括收集步骤3)离心后的脐带组织,重复使用1~3次。
7.根据权利要求1-6中任一项所述的方法从脐带组织中提取的复合细胞因子;
优选地,所述复合细胞因子包括干细胞因子(SCF)、基质细胞衍生因子-1(SDF-1)、血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、肝细胞生长因子(HGF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、胰岛素样生长因子(IGF)、白介素-6(IL-6)、白介素-8(IL-8)、单核细胞趋化蛋白-1(MCP-1)、金属蛋白酶移植因子(TIMP-1)。
8.一种保存如权利要求7所述的复合细胞因子的方法,包括将所述复合细胞因子分装于西林瓶中,在3h~6h内,冷冻到﹣50℃,抽真空干燥后,使温度上升到5±3℃,可长期保存备用。
9.一种使用如权利要求7所述的复合细胞因子的方法,包括将所述冻干的复合细胞因子加入2-4倍体积的灭菌用水溶解。
10.如权利要求1-6中任一项所述的方法提取的复合细胞因子或者如权利要求7所述的复合细胞因子在制备用于细胞培养、组织修复、医疗美容或生物3D打印的材料或组合物中的用途。
11.一种用于细胞培养、组织修复、医疗美容或生物3D打印的材料或组合物,其包含如权利要求1-6中任一项所述的方法提取的复合细胞因子或者如权利要求7所述的复合细胞因子。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109867718A (zh) * | 2019-04-11 | 2019-06-11 | 南京鼓楼医院 | 一种从神经干细胞来源的高浓度复合再生因子的制备方法及其用途 |
CN111728223A (zh) * | 2020-07-07 | 2020-10-02 | 西安因子元素科技有限公司 | 一种发酵生产复合细胞因子元素的方法 |
CN114652664A (zh) * | 2020-12-23 | 2022-06-24 | 科索瑞生物科技(天津)有限公司 | 修复凝胶及其制备方法 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104027794A (zh) * | 2014-06-19 | 2014-09-10 | 徐妍 | 人脐带间充质干细胞复合细胞因子在制备修复皮肤损伤的生物制剂中的应用 |
CN106344493A (zh) * | 2016-10-12 | 2017-01-25 | 领航干细胞再生医学工程有限公司 | 含人间充质干细胞因子的精华液的制备方法 |
CN106367386A (zh) * | 2016-10-14 | 2017-02-01 | 中卫华医(北京)生物科技有限公司 | 人脐带间充质干细胞因子冻干粉的制备方法 |
-
2017
- 2017-05-17 CN CN201710346450.XA patent/CN107236032B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104027794A (zh) * | 2014-06-19 | 2014-09-10 | 徐妍 | 人脐带间充质干细胞复合细胞因子在制备修复皮肤损伤的生物制剂中的应用 |
CN106344493A (zh) * | 2016-10-12 | 2017-01-25 | 领航干细胞再生医学工程有限公司 | 含人间充质干细胞因子的精华液的制备方法 |
CN106367386A (zh) * | 2016-10-14 | 2017-02-01 | 中卫华医(北京)生物科技有限公司 | 人脐带间充质干细胞因子冻干粉的制备方法 |
Non-Patent Citations (1)
Title |
---|
罗云波: "《食品商生物技术导论》", 31 August 2002, 中国农业大学出版社 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109867718A (zh) * | 2019-04-11 | 2019-06-11 | 南京鼓楼医院 | 一种从神经干细胞来源的高浓度复合再生因子的制备方法及其用途 |
CN111728223A (zh) * | 2020-07-07 | 2020-10-02 | 西安因子元素科技有限公司 | 一种发酵生产复合细胞因子元素的方法 |
CN114652664A (zh) * | 2020-12-23 | 2022-06-24 | 科索瑞生物科技(天津)有限公司 | 修复凝胶及其制备方法 |
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