CN114874992A - 一种用于减重的重组细胞 - Google Patents
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Abstract
本发明公开了一种用于减重的重组细胞，其为间充质干细胞或免疫细胞(T细胞、NK细胞或DC细胞)，通过慢病毒表达载体表达Interleukin‑27，该慢病毒表达载体中的Interleukin‑27的表达核苷酸序列如SEQ ID NO.01所示。本发明利用间充质干细胞(或T细胞)作为表达载体，构建重组间充质干细胞(或重组免疫细胞)，并回输细胞对肥胖症状进行治疗，可以持续分泌IL27，与直接以细胞因子IL27给药相比，可以降低给药频率，且给药期间IL27可以维持在比较稳定的作用浓度。
Description
技术领域
本发明属于重组功能细胞技术领域，具体涉及一种用于减重的重组细胞。
背景技术
肥胖是人体内过剩的能量转化为脂肪并积聚在体内的一种状态。脂肪组织能够分泌瘦素、脂联素、TNF-α、IL-6等细胞因子，引起身体性的慢性炎症，导致二型糖尿病和心血管疾病等代谢性疾病。减重的基本原则为少吃多动，即适当减少饮食，增加运动量，同时也有必要探索新的减重方式。
Interleukin-27(IL27)是隶属于IL-6/IL-12家族的细胞因子，由EBI3和p28两条多肽链通过二硫键连接构成的异源二聚体，由活化的抗原呈递细胞即单核巨噬细胞和树突状细胞产生。IL27可以促进Th1型免疫而增强机体免疫，也可以通过抑制Th17细胞的分化等来避免机体免疫过度，参与机体抵抗炎症和抗肿瘤等生理过程。研究表明(1)，肥胖人群血液中IL27水平偏低，而接受IL27治疗的肥胖小鼠体重明显减轻。棕色脂肪组织在体内持续产热，从而保持正常体重，维持正常的代谢。IL27直接靶向棕色脂肪组织，与脂肪细胞表面的IL27Ra(即WSX-1)结合，并激活p38MAPK-PGC-1a信号通路，促进产热相关蛋白UCP1的表达，从而达到减重的效果。人的IL27Ra与家鼠的IL27Ra同源性为69％，人IL27与鼠IL27同源性为75％，同源性较好。但是，研究表明(2)，直接注射细胞因子IL27后，IL27在体内存在的半衰期较短，小鼠腹腔注射IL27五天后，IL27血药浓度仅剩初始给药量的5％-10％，因此直接使用细胞因子IL27进行治疗需要比较频繁的给药。
发明内容
本发明目的在于克服现有技术缺陷，提供一种用于减重的重组细胞。
本发明的技术方案如下：
一种重组间充质干细胞，其通过所负载的慢病毒表达载体表达Interleukin-27，该慢病毒表达载体中的Interleukin-27的表达核苷酸序列如SEQ ID NO.01所示。
在本发明的一个优选实施方案中，所述慢病毒表达载体包括包装质粒和目的质粒，该目的质粒为负载有所述表达核苷酸序列的pCDH-CMV-MCS-EF1质粒。
上述重组间充质干细胞在制备减重组合物中的应用。
一种减重组合物，其有效成分包括上述重组间充质干细胞。
在本发明的一个优选实施方案中，其有效成分为上述重组间充质干细胞。
一种重组免疫细胞，其通过所负载的慢病毒表达载体表达Interleukin-27，该慢病毒表达载体中的Interleukin-27的表达核苷酸序列如SEQ ID NO.01所示。
在本发明的一个优选实施方案中，所述慢病毒表达载体包括包装质粒和目的质粒，该目的质粒为负载有所述表达核苷酸序列的pCDH-CMV-MCS-EF1质粒。
上述重组免疫细胞在制备减重组合物中的应用。
一种减重组合物，其有效成分包括上述重组免疫细胞。
在本发明的一个优选实施方案中，其有效成分为上述重组免疫细胞。
优选的，上述重组免疫细胞的中的免疫细胞为T细胞、NK细胞或DC细胞。
本发明的有益效果是：
1、本发明利用间充质干细胞(或免疫细胞)作为表达载体，构建重组间充质干细胞(或重组免疫细胞)，并回输细胞对肥胖症状进行治疗，可以持续分泌IL27，与直接以细胞因子IL27给药相比，可以降低给药频率，且给药期间IL27可以维持在比较稳定的作用浓度。
2、本发明取小鼠异体间充质干细胞，通过基因改造得到重组间充质干细胞后回输，回输细胞可以在20-40天内持续外分泌表达IL27。
3、本发明取小鼠异体T细胞，通过基因改造得到重组间T细胞回输，回输细胞产生记忆T细胞后，可以持续分泌Interleukin-27。给予肥胖小鼠MSC-exIL27稳转细胞尾静脉回输治疗一个疗程后，肥胖小鼠体重显著减轻。
附图说明
图1为本发明实施例1中的sp-EBI3-1inker-p28的结构示意图。
图2为本发明实施例2中的Western Blot实验结果图。
图3为本发明实施例3中的Western Blot实验结果图。
图4为本发明实施例4中的小鼠MSC的流式表型图。
图5为本发明实施例4中的小鼠体内IL27浓度时间梯度图。
图6为本发明实施例4中的MSC-exIL27治疗，三组小鼠体重变化图。
图7为本发明实施例5中的T-exIL27治疗，三组小鼠体重变化图。
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
实施例1质粒表达载体的构建
在自然状态下，Interleukin-27是由EBI3和p28两条多肽链通过二硫键连接构成的异源二聚体，EBI3必须和p28结合后才能被有效地分泌到细胞外发挥免疫作用。本发明设计了IL27的表达序列如图1所示的sp-EBI3-linker-p28，sp片段为外分泌定位的信号肽，具体核苷酸序列如下(SEQ ID NO.01)：
通过分子克隆将目的序列转入pCDH-CMV-MCS-EF1质粒，得到质粒表达载体pCDH-CMV-MCS-EF1-IL27。
实施例2 MSC-exIL27稳转细胞构建及体外功能检测
(1)293T细胞的慢病毒包装：在6孔板培养293T细胞，至其密度为80％左右时，换用OMEM培养。每孔加入10μL lippo2000，2μg目的质粒(实施例1制得的质粒表达载体pCDH-CMV-MCS-EF1-IL27)和2μg包装质粒，转染并孵育。12h后换新鲜培养液，用含10％FBS的DMEM培养。慢病毒包装后48h收集病毒上清，3500rpm离心10min，收集上清，使用超滤管进行病毒浓缩，浓缩后的病毒-80℃保存。
(2)间充质干细胞的感染：将间充质干细胞接种于6孔板中，至其密度为50％左右时，每孔加入等同于浓缩前1.5mL的病毒浓缩液，并用DMEM(10％FBS)稀释至1.5mL，加入1.5μL polybrene(终浓度为10μg/mL)，六孔板于2500rpm，37℃离心30min。在37℃、5％CO2条件下感染24h，之后换新鲜培养液(DMEM F/12，10％FBS)继续培养。
(3)感染48h后，MSC-exIL27稳转细胞换DMEM F/12原液培养24h，之后取培养液，离心(3500rpm，10min，4℃)后取上清。以BCA法测定MSC-exIL27稳转细胞上清中蛋白浓度，WB检测IL27的表达，结果如图2所示。
(4)经过预实验，确定本实施例构建的MSC-exIL27稳转细胞具有持续外分泌IL27的功能。
实施例3 T-exIL27稳转细胞构建及体外功能检测
(1)293T细胞的慢病毒包装：同实施例2。
(2)T细胞的感染：将T细胞在激活培养基中培养24h。取2×105个T细胞加入24孔板1个孔，再加入150μL病毒液和350μL T细胞完全培养液，并加入0.5μL polybrene(终浓度10μg/mL)，混匀。24孔板于1000G，37℃离心1h。在37℃、5％CO2条件下感染6h，弃病毒液，相同条件再次感染12h，之后全换液为T细胞完全培养液。
(3)感染48h后，T-exIL27稳转细胞换原液培养24h，之后取培养液，离心(3500rpm，10min，4℃)，离心后取上清。以BCA法测定T-exIL27稳转细胞上清中蛋白浓度，WB检测IL27的表达，结果如图3所示。
(4)经过预实验，确定本实施例构建的T-exIL27稳转细胞具有持续外分泌IL27的功能。
实施例4 MSC-exIL27稳转细胞的小鼠实验
(1)构建小鼠肥胖模型：购买C57/BL6小鼠15只，喂食高脂鼠粮12周，每天每只小鼠20g高脂鼠粮，期间小鼠正常饲养。
(2)取小鼠外周血中间充质干细胞：戊巴比妥钠腹腔注射以全麻一只小鼠，麻醉后从心脏穿刺抽出血液约1.5mL，注入预先肝素化抗凝的干燥试管中，再用PBS按1∶1稀释。另取一离心管，管内注入Percoll分离液，将稀释好的血样按1∶1比例缓慢沿试管壁加入Percoll分离液。2000r/min离心20min，离心后上清分为四层，吸取最上层(血浆层)弃之，吸出第二层(单核细胞层)并注入另一个干燥离心管中，加入等量DMEM F/12原液，1500r/min离心10min，弃上清，重复洗涤1次。以1×106/mL密度接种于25cm2培养瓶中，加入DMEM F/12(10％FBS)培养液30mL，于37℃、5％CO2 -培养。48-72h首次半量换液，之后每4天全量换液1次，每次换液用PBS洗涤2次，此时细胞为P0代MSC。
传代培养：待细胞融合率达到80％左右，按1∶2进行传代培养，此时细胞称为P1代MSC，使用P1代MSC构建MSC-exIL27稳转细胞。流式检测P1代MSC表型，结果如图4所示。
(3)构建MSC-exIL27稳转细胞，检测表达：具体如实施例2所述，异体MSC-exIL27稳转细胞构建后进行扩培，扩培至一定数量的细胞进行回输。
(4)细胞治疗：将肥胖小鼠随机分为4组，分别尾静脉注射PBS、IL27、异体MSC、异体MSC-exIL27稳转细胞给予治疗，治疗周期为1个月，每周治疗一次，每次剂量为每只小鼠100万细胞，PBS注射相同体积，IL27按100μg/kg给药。每五天进行尾静脉取血1次，WB检测小鼠外周血中IL27水平(结果如图5所示)，并进行1次体重称量。如图6所示，治疗后，MSC-exIL27稳转细胞组小鼠体重显著下降。
实施例5 T-exIL27稳转细胞的小鼠实验
(1)构建小鼠肥胖模型：购买C57/BL6小鼠15只，喂食高脂鼠粮12周，每天每只小鼠20g高脂鼠粮，期间小鼠正常饲养。
(2)取小鼠脾脏T细胞：取一只小鼠脱颈椎处死，在75％酒精中浸泡片刻，剪开小鼠腹腔，取出小鼠脾脏，剪成小组织块后放置于尼龙网上，用注射器针栓截面仔细研磨组织块，随后用生理盐水冲洗和收集，不断重复这个过程，直到脾脏碎块体积不再变化。
取市售小鼠淋巴细胞分离液，15mL试管加入3mL，将收集的细胞悬液沿管壁轻轻加到分离液上，1800rpm离心20min，离心后自上而下分为5层，小心吸取单核细胞层移入另一试管，PBS洗涤两次。
流式检测小鼠脾脏淋巴细胞表型。
(3)构建异体T-exIL27稳转细胞，检测表达。具体如实施例3所述，异体T-exIL27稳转细胞构建后进行扩培，扩培至一定数量的细胞进行回输。
(4)细胞治疗：将肥胖小鼠随机分为4组，分别尾静脉注射PBS、IL27、异体T细胞、异体T-exIL27给予治疗，治疗周期为1个月，每周治疗一次，每次剂量为每只小鼠100万细胞，PBS注射相同体积，IL27按100μg/kg给药。每五天进行尾静脉取血1次，WB检测小鼠外周血中IL27水平，并进行1次体重称量。如图7所示，治疗后，T-exIL27稳转细胞组小鼠体重显著下降。
以上所述，仅为本发明的较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。
序列表
<110> 厦门星际诺康细胞科技有限公司
<120> 一种用于减重的重组细胞
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1401
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gaattcgccg ccaccatgga cgccatgaag agaggcctgt gctgcgtgct gctgctgtgc 60
ggcgctgtgt ttgtgagccc tagaaagggg cctcctgccg ctctgaccct gcctagagtg 120
cagtgcagag ccagcaggta tcccatcgcc gtcgattgca gctggaccct gccccctgct 180
cccaacagca catctcccgt gagcttcatc gccacctaca gactgggaat ggctgccaga 240
ggccacagct ggccctgcct gcagcagaca cccacttcta caagctgcac catcaccgac 300
gtgcagctgt tcagcatggc tccctacgtg ctgaacgtga ccgccgtgca cccctggggc 360
tctagctcta gcttcgtgcc cttcatcacc gagcacatca tcaagcccga ccctcccgag 420
ggcgtgaggc tgtctcctct ggccgagaga cagcttcaag tacagtggga acctcccggc 480
agctggccct tccccgagat cttcagcctg aagtactgga tcagatacaa gagacaaggc 540
gctgccaggt tccacagagt gggccctatc gaggccacaa gcttcatcct gagagccgtc 600
agacctagag ctagatacta cgtgcaagtg gctgcccaag acctcaccga ctacggcgag 660
ttaagcgatt ggtctctgcc tgccaccgca accatgtcat taggcaaagg aggcgggagc 720
ggcggtgggt ctggcggcgg aagctttcct agacctcccg gcagacctca gctgagcctg 780
caagagctga ggagagagtt caccgtgtct ctgcacctgg ctagaaagct gctgagcgag 840
gtgagaggcc aagcccacag gttcgccgag agccacctgc ctggcgtgaa cctgtacctg 900
ctgcctctgg gcgagcagct gcccgacgtg agcctgacct tccaagcctg gaggagactg 960
agcgaccccg agagactgtg cttcatctct accactctgc agcccttcca cgccctgctg 1020
ggcggcttag gtacccaagg cagatggacc aacatggaga gaatgcagct gtgggccatg 1080
aggctggacc tgagagacct gcagagacac ctgagattcc aagtgctggc tgctggcttc 1140
aacctgcccg aggaggagga ggaggaggag gaggaggaag aggaggaaag aaagggactg 1200
ctccctggtg ccttaggctc tgcactacaa ggccctgccc aagtgagctg gcctcagctg 1260
ctgtctacct acaggttgct gcacagcctg gagctggtgc tgagcagagc agtgagggaa 1320
ctgctcctcc tgagcaaggc tgggcacagc gtgtggcctc tgggcttccc cactctgagc 1380
cctcagccct gataaggatc c 1401
Claims (10)
1.一种重组间充质干细胞，其特征在于：其通过所负载的慢病毒表达载体表达Interleukin-27，该慢病毒表达载体中的Interleukin-27的表达核苷酸序列如SEQ IDNO.01所示。
2.如权利要求1所述的一种重组间充质干细胞，其特征在于：所述慢病毒表达载体包括包装质粒和目的质粒，该目的质粒为负载有所述表达核苷酸序列的pCDH-CMV-MCS-EF1质粒。
3.权利要求1或2所述的重组间充质干细胞在制备减重组合物中的应用。
4.一种减重组合物，其特征在于：其有效成分包括权利要求1或2所述的重组间充质干细胞。
5.如权利要求4所述的一种减重组合物，其特征在于：其有效成分为权利要求1或2所述的重组间充质干细胞。
6.一种重组免疫细胞，其特征在于：其通过所负载的慢病毒表达载体表达Interleukin-27，该慢病毒表达载体中的Interleukin-27的表达核苷酸序列如SEQ IDNO.01所示。
7.如权利要求6所述的一种重组免疫细胞，其特征在于：所述慢病毒表达载体包括包装质粒和目的质粒，该目的质粒为负载有所述表达核苷酸序列的pCDH-CMV-MCS-EF1质粒。
8.权利要求6或7所述的重组免疫细胞在制备减重组合物中的应用。
9.一种减重组合物，其特征在于：其有效成分包括权利要求6或7所述的重组免疫细胞。
10.如权利要求9所述的一种减重组合物，其特征在于：其有效成分为权利要求6或7所述的重组免疫细胞。
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