CN111840643A - 一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法 - Google Patents
一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法 Download PDFInfo
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Abstract
本发明涉及一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,包括以下步骤:1)将抽取的脂肪组织混合液置入试管内,离心后吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;2)加入核酸型发酵液内,再加入生理盐水搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使用半导体制冷设备冷冻;3)用冰冻离心机进行离心作业,在发酵液重新液化后,静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理;4)离心过滤后,放入冰冻离心机进行离心作业,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得活性因子;本发明可促进原有的脂肪细胞继续生长,最终稳定成为脂肪组织,且成活率高。
Description
[技术领域]
本发明涉及生物细胞技术领域,具体地说是一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法。
[背景技术]
脂肪主要分布在人体皮下组织、大网膜、肠系膜和肾周等部位。脂肪组织是由前脂肪细胞和成熟脂肪细胞构成,成熟脂肪细胞中的包浆中含有90%的脂滴,其移植后可迅速代谢掉。成熟脂肪细胞是分化终末细胞,已经失去了继续增殖的能力。成熟脂肪细胞只能使移植部位的体积增大且加上成熟脂肪细胞易受外界机械损伤,而使得相当大部分成熟脂肪细胞受到损伤而破坏。常用的脂肪移植的是经过处理的脂肪颗粒,去除油脂或血水等杂质,移植后会部分坏死,成活率在30%~70%左右。
自体脂肪移植技术已经广泛地应用于丰胸、隆鼻或者疤痕修复等整形手术中。或者通过注射的方式再移植到自己需要进行脂肪填充的部位,例如乳房、面部等,用以治疗胸部扁平、两侧乳房不对称、浅表微细皱纹、薄嘴唇隆成厚嘴唇等。虽然自体脂肪相较于传统的人工组织替代物具有相容性好、操作简便、填充外形好、手术创伤小、来源丰富等优势。然而,目前很多脂肪填充后有一定程度的被吸收,为了保证最好的效果需要进行再次手术,脂肪的用量过多或注射过于集中,大量脂肪堆积会因供血不足导致脂肪坏死、溶解、吸收,极易引发感染,出现纤维化或钙化、脂肪坏死等后遗症,很多的自体脂肪在注射之后,成活率都较低,会造成手术的负面效果,影响手术成果。
[发明内容]
本发明的目的就是要解决上述的不足而提供一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,可促进原有的脂肪细胞继续生长,最终稳定成为脂肪组织,且成活率高,解决了传统脂肪移植吸收活性脂肪细胞移植吸收或坏死等问题。
为实现上述目的设计一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,包括以下步骤:1)在无菌条件下,将抽取的脂肪组织混合液置入试管内,在离心机内离心处理15min,离心机转速为1200rpm,使得全血样本初步分离,离心后血液加入抗凝剂,静置30-60分钟,将油脂层弃去,吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;2)将步骤1)所得的样本加入核酸型发酵液内,得到样本发酵液,加入生理盐水搅拌,搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使发酵液上清与冻存液混匀后,加入冻存容器,使用半导体制冷设备冷冻,半导体制冷设备温度降低速率为10-12℃/min;3)将上一步骤冷冻的发酵液取出并消化成黏糊状,用冰冻离心机进行离心作业,经生理盐水水浴,生理盐水浓度为5%,在发酵液重新液化后,静置处理,并将静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理,离心机转速保持1200rpm;4)离心完成后,在离心管内加入生理盐水,在恒温环境下静置处理,恒温温度为22-24℃,用滤网过滤后,放入冰冻离心机进行离心作业,弃掉离心管上层的血浆,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得到活性因子。
进一步地,步骤1)中,所述抗凝剂与全血样本比例为1:10-12,所述抗凝剂采用乙二胺四乙酸、草酸钠、肝素与枸橼酸钠混合制成。
进一步地,步骤2)中,所述冻存液为小牛血清与DMSO混合物;优选为,所述小牛血清与DMSO比例为1:8
进一步地,步骤4)中,在离心管内所加入的生理盐水浓度为6%,用70μm滤网过滤后,放入冰冻离心机以1200r/min,离心6min。
进一步地,步骤4)之后,将所得到的活性因子接种于培养皿内,置于36℃,5%CO2培养箱内孵育,隔日换液,之后每日观察,每3-4天换液一次。
本发明同现有技术相比,具有如下优点:
(1)本发明具有完善的血液循环和营养供给功能,移植后可不断分化成脂肪的细胞,解决了传统脂肪的移植吸收活性脂肪细胞移植吸收或坏死等问题,可使移植的脂肪长期、持久、稳定的存活;
(2)本发明促进了原有脂肪细胞继续生长,可帮助形成血管,加速移植脂肪的血管化和再成活,可释放促进血管形成因子,可使移植的脂肪快速成活为正常的、有生命力的脂肪组织;
(3)本发明可显著提升患者形体美感,是一种显著优于传统单纯脂肪组织移植过程的提升患者形体美的方法;
(4)本发明提取的材料移植后转化为脂肪细胞,可促进原有的脂肪细胞继续生长,最终稳定成为脂肪组织,成活率高达90%以上;
(5)本发明能够保证自体脂肪注射后的活力,使得自体脂肪注射手术更加安全可靠,值得推广。
[具体实施方式]
下面结合具体实施例对本发明作以下进一步说明:
实施例1
一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,包括以下步骤:
1)在无菌条件下,将抽取的脂肪组织混合液置入试管内,在离心机内离心处理15min,离心机转速为1200rpm,使得全血样本初步分离,离心后血液加入抗凝剂,静置30-60分钟,将油脂层弃去,吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;其中,抗凝剂与全血样本比例为1:10,抗凝剂采用乙二胺四乙酸、草酸钠、肝素与枸橼酸钠混合制成;
2)将步骤1)所得的样本加入核酸型发酵液内,得到样本发酵液,加入生理盐水搅拌,搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使发酵液上清与冻存液混匀后,加入冻存容器,使用半导体制冷设备冷冻,半导体制冷设备温度降低速率为10-12℃/min;
3)将上一步骤冷冻的发酵液取出并消化成黏糊状,用冰冻离心机进行离心作业,经生理盐水水浴,生理盐水浓度为5%,在发酵液重新液化后,静置处理,并将静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理,离心机转速保持1200rpm;
4)离心完成后,在离心管内加入生理盐水,在恒温环境下静置处理,恒温温度为22-24℃,用滤网过滤后,放入冰冻离心机进行离心作业,弃掉离心管上层的血浆,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得到活性因子。
实施例2
一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,包括以下步骤:
1)在无菌条件下,将抽取的脂肪组织混合液置入试管内,在离心机内离心处理15min,离心机转速为1200rpm,使得全血样本初步分离,离心后血液加入抗凝剂,静置30-60分钟,将油脂层弃去,吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;其中,抗凝剂与全血样本比例为1:12,抗凝剂采用乙二胺四乙酸、草酸钠、肝素与枸橼酸钠混合制成;
2)将步骤1)所得的样本加入核酸型发酵液内,得到样本发酵液,加入生理盐水搅拌,搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使发酵液上清与冻存液混匀后,加入冻存容器,使用半导体制冷设备冷冻,半导体制冷设备温度降低速率为10-12℃/min;其中,冻存液为小牛血清与DMSO混合物,小牛血清与DMSO比例为1:8;
3)将上一步骤冷冻的发酵液取出并消化成黏糊状,用冰冻离心机进行离心作业,经生理盐水水浴,生理盐水浓度为5%,在发酵液重新液化后,静置处理,并将静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理,离心机转速保持1200rpm;
4)离心完成后,在离心管内加入生理盐水,在恒温环境下静置处理,恒温温度为22-24℃,用滤网过滤后,放入冰冻离心机进行离心作业,弃掉离心管上层的血浆,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得到活性因子;其中,在离心管内所加入的生理盐水浓度为6%,用70μm滤网过滤后,放入冰冻离心机以1200r/min,离心6min。
实施例3
一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,包括以下步骤:
1)在无菌条件下,将抽取的脂肪组织混合液置入试管内,在离心机内离心处理15min,离心机转速为1200rpm,使得全血样本初步分离,离心后血液加入抗凝剂,静置30-60分钟,将油脂层弃去,吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;其中,抗凝剂与全血样本比例为1:10,抗凝剂采用乙二胺四乙酸、草酸钠、肝素与枸橼酸钠混合制成;
2)将步骤1)所得的样本加入核酸型发酵液内,得到样本发酵液,加入生理盐水搅拌,搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使发酵液上清与冻存液混匀后,加入冻存容器,使用半导体制冷设备冷冻,半导体制冷设备温度降低速率为10-12℃/min;
3)将上一步骤冷冻的发酵液取出并消化成黏糊状,用冰冻离心机进行离心作业,经生理盐水水浴,生理盐水浓度为5%,在发酵液重新液化后,静置处理,并将静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理,离心机转速保持1200rpm;
4)离心完成后,在离心管内加入生理盐水,在恒温环境下静置处理,恒温温度为22-24℃,用滤网过滤后,放入冰冻离心机进行离心作业,弃掉离心管上层的血浆,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得到活性因子;
5)将所得到的活性因子接种于培养皿内,置于36℃,5%CO2培养箱内孵育,隔日换液,之后每日观察,每3-4天换液一次。
本发明并不受上述实施方式的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于,包括以下步骤:
1)在无菌条件下,将抽取的脂肪组织混合液置入试管内,在离心机内离心处理15min,离心机转速为1200rpm,使得全血样本初步分离,离心后血液加入抗凝剂,静置30-60分钟,将油脂层弃去,吸取最上层的上清血浆,加入羟乙基淀粉溶液充分混匀;
2)将步骤1)所得的样本加入核酸型发酵液内,得到样本发酵液,加入生理盐水搅拌,搅拌后静置,用无菌注射器抽取发酵液上清,并在发酵液上清中加入冻存液,使发酵液上清与冻存液混匀后,加入冻存容器,使用半导体制冷设备冷冻,半导体制冷设备温度降低速率为10-12℃/min;
3)将上一步骤冷冻的发酵液取出并消化成黏糊状,用冰冻离心机进行离心作业,经生理盐水水浴,生理盐水浓度为5%,在发酵液重新液化后,静置处理,并将静置后的液体加入离心管,将离心管放在离心机内进行二次离心处理,离心机转速保持1200rpm;
4)离心完成后,在离心管内加入生理盐水,在恒温环境下静置处理,恒温温度为22-24℃,用滤网过滤后,放入冰冻离心机进行离心作业,弃掉离心管上层的血浆,加入新的血浆样本,使得沉淀的血小板重新悬浮,即得到活性因子。
2.如权利要求1所述的提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于:步骤1)中,所述抗凝剂与全血样本比例为1:10-12,所述抗凝剂采用乙二胺四乙酸、草酸钠、肝素与枸橼酸钠混合制成。
3.如权利要求1所述的提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于:步骤2)中,所述冻存液为小牛血清与DMSO混合物。
4.如权利要求3所述的提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于:所述小牛血清与DMSO比例为1:8。
5.如权利要求1所述的提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于:步骤4)中,在离心管内所加入的生理盐水浓度为6%,用70μm滤网过滤后,放入冰冻离心机以1200r/min,离心6min。
6.如权利要求1所述的提高自体脂肪活细胞等移植性自体脂肪成活率的活性因子的萃取方法,其特征在于:步骤4)之后,将所得到的活性因子接种于培养皿内,置于36℃,5%CO2培养箱内孵育,隔日换液,之后每日观察,每3-4天换液一次。
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