CN106544316B - 一种牙髓干细胞膜片的制备方法 - Google Patents
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Abstract
本发明实施例提供了一种牙髓干细胞膜片的制备方法,包括从牙髓组织中分离出干细胞;将分离出的干细胞接种于细胞培养基中培养,其中,细胞培养基的配方为基础培养基添加转化生长因子β1和白介素10;将培养后的干细胞制备成细胞膜片。该方法既降低了牙髓干细胞膜片的免疫原性,又增强了牙髓干细胞膜片的免疫抑制性。
Description
技术领域
本发明涉及医学及细胞生物技术领域,具体涉及一种牙髓干细胞膜片的制备方法。
背景技术
牙周炎是口腔科临床的常见病,是以牙周组织破坏为主要特征的感染性疾病,而一旦牙周附着和牙槽骨遭到破坏,就可能表现为牙龈炎症和出血,牙周袋的形成,牙槽骨吸收,牙齿松动甚至丧失。牙周炎不仅是牙齿丧失的主要原因,而且与一些全身系统性疾病的发生发展有关,比如糖尿病,心血管疾病等。牙周炎丧失的牙周组织尤其是骨组织,其修复与重建的难度很大,所以牙周炎的治疗一直是口腔临床研究的重点和难点。
发明内容
本发明的实施例提供了一种牙髓干细胞膜片的制备方法,该方法既降低了牙髓干细胞膜片的免疫原性,又增强了牙髓干细胞膜片的免疫抑制性。
为达到上述目的,本发明的实施例采用了牙髓干细胞膜片的制备方法,包括从牙髓组织中分离出干细胞;将分离出的干细胞接种于细胞培养基中培养,其中,所述细胞培养基的配方为基础培养基添加转化生长因子β1和白介素10(interleukin-10,IL-10);将培养后的干细胞制备成细胞膜片。
可选地,所述的基础培养基为α-MEM液体培养基。
可选地,所述将培养后的干细胞制备成细胞膜片的步骤具体为:将培养后的干细胞接种于加入了基质金属蛋白酶9中和抗体的培养基;待处于所述培养基边缘处的干细胞出现皱褶时,将细胞膜片揭下。
可选地,所述转化生长因子β1的浓度为30ng/ml-150ng/ml。
可选地,所述白介素10的浓度为10ng/ml-100ng/ml。
可选地,所述基质金属蛋白酶9中和抗体的浓度1μg/ml-10μg/ml。
可选地,制备成的细胞膜片包括1-6层牙髓干细胞DPSCs。
可选地,所述从牙髓组织中分离出干细胞的步骤具体为:从健康的第三磨牙或前磨牙的牙髓组织中分离出干细胞。
可选地,所述将培养后的干细胞制备成细胞膜片之前,还包括:对所述培养后的干细胞进行鉴定。
可选地,对所述培养后的干细胞进行鉴定的步骤包括:检测所述培养后的干细胞是否能够进行骨向诱导分化,并检测所述培养后的干细胞是否能够进行脂肪向诱导分化。
本申请在将牙髓干细胞(Dental Pulp Stem Cells,DPSCs)制作成细胞膜片的过程中,在培养DPSCs的培养基中添加了两种免疫调节因子-转化生长因子β1和IL-10,由于这两种免疫调节因子既能够抑制混合淋巴细胞反应引起的T淋巴细胞增殖和植物血凝素(Phytohaemagglutinin,PHA)引起的T淋巴细胞增殖,以及抑制B淋巴细胞增殖,又能够抑制炎性细胞因子的分泌和T细胞表面的抗原表达,并诱导免疫耐受,因此,在基础培养基的基础上,加入这两种免疫调节因子,使得制备而成的牙髓干细胞膜片既具有低免疫原性,又具有较强的免疫抑制性。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为一种牙髓干细胞膜片的制备方法流程图;
图2a为DPSCs标志物STRO-1的表达示意图;
图2b为DPSCs标志物CD146的表达示意图;
图2c为DPSCs标志物CD90的表达示意图;
图3为DPSCs骨向诱导分化的示意图;
图4为DPSCs脂肪向诱导分化的示意图;
图5a为DPSCs不会引起PBMCs增殖的示意图;
图5b为DPSCs抑制PHA引起的T淋巴细胞增殖的示意图;
图5c为DPSCs抑制混合细胞淋巴反应引起的T淋巴细胞增殖的示意图;
图5d为DPSCs抑制B淋巴细胞增殖的示意图;
图6a为DPSCs对IL-2细胞因子分泌的影响示意图;
图6b为DPSCs对IL-17细胞因子分泌的影响示意图;
图6c为DPSCs对IFN-γ细胞因子分泌的影响示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
目前,临床上有应用牙周膜干细胞治疗牙周炎的方法,但是这种方法存在一定的局限性:因为牙齿的牙周膜组织比较少,质地比较韧,所以分离以及培养牙周膜干细胞比较困难。而本申请的发明人发现,牙齿的牙髓组织比较疏松,组织量多,分离培养DPSCs比较容易。但是考虑到在治疗牙周炎时,往往需要异体移植DPSCs或者牙髓干细胞膜片到受体中,所以免疫排斥反应是一个必须要考虑的问题。基于上述考虑,本发明提供了一种牙髓干细胞膜片的制备方法,该方法在培养DPSCs的培养基中添加了转化生长因子β1和IL-10(interleukin-10,IL-10)这两种免疫调节因子,由于这两种免疫调节因子既能够抑制混合淋巴细胞反应引起的T淋巴细胞增殖和PHA引起的T淋巴细胞增殖,以及抑制B淋巴细胞增殖,又能够抑制炎性细胞因子的分泌和T细胞表面的抗原表达,并诱导免疫耐受,因此,在基础培养基的基础上,加入这两种免疫调节因子,使得制备而成的牙髓干细胞膜片既具有低免疫原性,又具有较强的免疫抑制性,进而降低了在异体移植细胞膜片时出现免疫排斥反应的可能性。
本发明方案在具体实现时,如图1所示,首先进行步骤S1:从牙髓组织中分离出干细胞。步骤如下:将拔除的牙齿放入无菌且有预冷磷酸缓冲盐溶液(Phosphate BufferSaline,PBS)的离心管,并移送至细胞室,将牙齿纵向劈开,取出牙髓组织,用PBS反复清洗,剪碎,置于含I型胶原酶(3g/L)和中性蛋白酶(4g/L)的消化液中,在37℃的温度下消化1小时后,用70μm的细胞筛收集细胞,并以1000转/分钟离心10分钟,用培养液重新悬浮成单细胞悬液。
完成步骤S1后,执行步骤S2:将分离出的干细胞接种于细胞培养基中培养。具体操作为:将分离出的干细胞接种于25㎝2的细胞培养瓶中,在添加了转化生长因子β1和IL-10的基础培养基中37℃、5%CO2培养,此处的基础培养基为α-MEM培养基,其成分包含15%胎牛血清、100μmol/L的L-抗坏血酸2-磷酸、2mmol/L谷氨酰胺、100U/ml青霉素和100μg/ml链霉素。每2-3天换液1次。每天在倒置显微镜下观察细胞生长状况。当细胞生长至80%汇合状态时,用0.25%胰蛋白酶按1:2消化传代。
可选地,转化生长因子β1的浓度为30ng/ml-150ng/ml,优选为100ng/ml。
可选地,IL-10的浓度为10ng/ml-100ng/ml,优选为50ng/ml。
可选地,取健康的第三磨牙或前磨牙为原料,分离和培养DPSCs。因为牙周炎在中老年人群中发病率较高,但是患有牙周炎的中老年人自身的DPSCs有限,所以如果取牙周炎患者的健康牙齿来分离和培养DPSCs会得不偿失。考虑到一些青少年在进行牙齿矫正时,一般会拔除作为阻生牙的第三磨牙,以及拔除前磨牙来开拓间隙,目前,这些被拔掉的第三磨牙和前磨牙作为医疗废物被扔掉了。因此,我们可以利用这些健康的第三磨牙或前磨牙来分离和培养DPSCs。如此,既充分利用了医疗资源,又扩大了DPSCs的来源。
可选地,在将DPSCs进行分离培养之后,还包括对培养后的DPSCs进行鉴定,其鉴定方法为:
首先取一部分培养的细胞,检测其骨向诱导分化能力和脂肪向诱导分化能力,即首先检测所培养的细胞是否为干细胞。
检测所培养细胞是否具有骨向诱导分化能力的步骤为:将所培养的细胞放入骨向诱导分化培养基中,这里的骨向诱导分化培养基除了常规细胞培养的基础培养基外,还需加10mmol/Lβ-甘油磷酸钠、10nmol/L地塞米松和50mg/L维生素C。在骨向诱导分化培养基中培养四周后,用Von-kossa染色法检测钙质沉积情况。具体步骤如下:将诱导后的细胞用PBS洗3次;95%的酒精固定30分钟;蒸馏水冲洗5分钟;用2%硝酸银溶液于紫外光下还原20分钟;用蒸馏水充分冲洗;用5%硫代硫酸钠作用3分钟,终止反应;显微镜下观察Von-kossa钙质染色结果。若Von-kossa钙质染色呈现较多的棕褐色阳性反应区域,该棕褐色阳性反应区域如图3中的深色区域显示,则说明所培养的细胞能够进行骨向诱导分化。
检测所培养细胞是否具有脂肪向诱导分化能力的步骤为:将所培养的细胞放入脂肪向诱导分化培养基中,这里的脂肪向诱导分化培养基除了常规细胞培养的基础培养基外,还需加0.5mmol/L异丁基-甲基黄嘌呤、60μmol/L消炎痛、0.5μmol/L氢化可的松和10μg/L胰岛素。
在上述培养基培养四周后,用油红染色方法观察脂肪细胞的形成情况,具体操作时,细胞诱导后用PBS洗两次;4%多聚甲醛室温下固定10分钟;再用PBS洗两次,然后用60%的异丙醇浸洗一次;室温下用油红O工作液染色30分钟;之后,再用60%的异丙醇浸洗一次,PBS洗两次;最后,放在显微镜下观察甘油三酯脂滴被染色的情况。若发现细胞内脂滴聚集,并呈现橙红色染色,则说明所培养的细胞能够进行脂肪向诱导分化,该橙红色染色用图4中的深色区域显示。
上述经过骨向诱导分化和脂肪向诱导分化检测,证明所培养的细胞为干细胞。
可选地,还可以通过检测标志物的表达情况进一步地鉴定所培养的细胞为DPSCs,具体操作步骤为:
用4%多聚甲醛室温固定DPSCs20分钟;
用PBS洗一次,再用PBS混悬细胞,调整细胞浓度为105个细胞/200μl;
取200μl细胞混悬液,分别加入STRO-1抗体(1:100)、CD146抗体(1:200)和CD90抗体(1:100),室温下反应1小时;
1000转/分钟离心5分钟,用PBS洗一次;
弃上清,加入100μlPBS混悬细胞;
加入异硫氰酸荧光素FITC(Fluorescein Isothiocyanate)标记的抗小鼠IgG抗体(1:200),室温避光30分钟。流式细胞仪上样,检测表达情况。
若如图2a所示,标志物STRO-1被表达,图2b所示,标志物CD146被表达,图2c所示,标志物CD90被表达,则可以证明上述培养的干细胞为DPSCs。
因为上面已经讲过,患有牙周炎的患者一般为中老年人,但是DPSCs的主要来源—健康的第三磨牙或者前磨牙主要来自青少年,因此将来自青少年的第三磨牙或者前磨牙的DPSCs移植至中老年患者缺损的牙周骨组织时,很可能会产生异体排斥反应。因此,还要取一部分干细胞进行免疫学检测。检测步骤为:
首先检测DPSCs免疫原性,具体操作时,将DPSCs(5.0×104)铺板贴壁,加入等量的异体T淋巴细胞,在37℃、5%CO2条件下培养5天。并以5.0×104单纯淋巴细胞培养、来自两个不同个体的5.0×104T淋巴细胞反应作为对照。用CCK-8试剂盒来检测异体T淋巴细胞的增殖情况。
需要说明的是,这里的T淋巴细胞可以为外周血单个核细胞(Peripheral BloodMononuclear Cells,PBMCs)。如图5a所示,DPSCs不会引起PBMCs增殖。
进一步地,检测DPSCs的免疫抑制性。免疫抑制性的检测可以包括DPSCs对PHA引起的T淋巴细胞增殖的影响,对混合淋巴细胞反应引起的T淋巴细胞增殖的影响,以及对B淋巴细胞增殖的影响。下面依次对这三种检测进行描述。
检测DPSCs对PHA引起的T淋巴细胞增殖的影响,具体操作时,将不同量的DPSCs(2.5×104、5.0×104或者2.5×105)铺板贴壁后,加入5.0×104的PBMCs(DPSCs:PBMCs分别为0.2:1、1:1、5:1)和终浓度为0.5μg/mL的PHA在37℃、5%CO2条件下培养5天。同法用CCK-8试剂盒来检测T淋巴细胞的增殖情况。
在本申请中,如图5b所示,DPSCs能够抑制PHA引起的T淋巴细胞增殖。
检测DPSCs对混合淋巴细胞反应引起的T淋巴细胞增殖的影响。具体操作时,将不同量的DPSCs(2.5×104、5.0×104或者2.5×105)铺板贴壁后,加入另外两个个体的异体PBMCs(细胞量为5.0×104),在37℃、5%CO2条件下培养5天。同法用CCK-8试剂盒来检测T淋巴细胞的增殖情况。
在本申请中,如图5c所示,DPSCs能够抑制混合细胞淋巴反应引起的T淋巴细胞增殖。
检测DPSCs对B淋巴细胞增殖的影响,具体操作时,将不同量的DPSCs(2.5×104、5.0×104或者2.5×105)铺板贴壁后,加入5.0×104的B淋巴细胞(DPSCs:B淋巴细胞分别为0.2:1、1:1、5:1)和B淋巴细胞增殖刺激因子,在37℃、5%CO2条件下培养5天。同法用CCK-8试剂盒来检测B淋巴细胞的增殖情况。
在本申请中,如图5d所示,DPSCs能够抑制B淋巴细胞的增殖。
为了进一步检测DPSCs的免疫抑制特性,本申请还对可溶性因子进行了检测,步骤为:
应用酶联免疫吸附测定法(Enzyme Linked Immunosorbent Assay,ELISA)测定PBMCs(5.0×104)+PHA(0.5μg/mL)+DPSCs(5.0×104)上清中的IL-2、IL-17、IFN-γ的浓度。
将5.0×104的DPSCs接种于24孔培养板中,在37℃、5%CO2条件下培养2个小时,使细胞贴壁。然后加入等量的异体PBMCs和终浓度为0.5μg/mL的PHA。在37℃、5%CO2条件下培养5天。5天后,吸取培养上清至5ml离心管中。1000转/分钟离心10分钟后,吸取上清至新的离心管,置于-80℃冰箱冻存。以单纯淋巴细胞(5.0×104)、PBMCs(5.0×104)+PHA(0.5μg/mL)作为对照。以IL-2、IL-17、IFN-γ标准品吸光度值绘制标准曲线,并建立回归方程后求出待检测标本中IL-2、IL-17、IFN-γ的浓度。
在本申请中,如图6a所示为所测炎性细胞因子IL-2的浓度变化图,图6b所示为所测炎性细胞因子IL-17的浓度变化图,图6c所示为所测炎性细胞因子IFN-γ的浓度变化图。从上述三个图中可以看出,DPSCs能显著下调IL-2,IL-17,IFN-γ的浓度,这就说明DPSCs对炎性细胞因子有抑制作用,进一步说明DPSCs具有免疫抑制性。
上面的描述证明了经过步骤S2培养后的DPSCs具有低免疫原性以及免疫抑制性。
接下来可以进行步骤S3:将培养后的干细胞制备成细胞膜片。具体操作时,将生长旺盛的第二代DPSCs(5.0×106)接种在培养成分为α-MEM培养基(含15%胎牛血清,2mmol/L谷氨酰胺,100U/ml青霉素,100μg/ml链霉素)的60mm培养皿中。为获得完整的细胞膜片,将20.0μg/ml的维生素C加入α-MEM培养基,进一步地,为了使得所制备的细胞膜片具有更好的机械强度和坚韧的质地,本申请在制备细胞膜片的培养基中还加入了基质金属蛋白酶9中和抗体,并在加入了该中和抗体的培养基中培养10-14天,待处于培养皿边缘处的细胞出现皱褶时,用细胞刮将细胞膜片整体揭下。
可选地,上述的细胞膜片为1-6层DPSCs,优选为3-4层DPSCs。基质金属蛋白酶9中和抗体的浓度为1μg/ml-10μg/ml,优选为5μg/ml。
因为制备细胞膜片的培养基中很有可能含有基质金属蛋白酶9,而基质金属蛋白酶9可以降解细胞外基质,进而使得细胞与细胞之间的连接不够紧密,导致所制得的细胞膜片质地松软,强度较低。在本申请中,在制备细胞膜片的培养基中加入了基质金属蛋白酶9中和抗体,就减弱了基质金属蛋白酶9的降解作用,因而使得细胞与细胞之间的连接更加紧密,在这种培养基中所制备的细胞膜片也就具有较高的机械强度和坚韧的质地,容易成型,而且,通过免疫检测实验,使用本申请的方法制备而成的细胞膜片消化成细胞后,同样具有低免疫原性和免疫抑制性,因此,在移植这种牙髓干细胞膜片至受体时,既降低了异体移植时产生免疫排斥反应的可能性,又可以不使用支架材料作为辅助工具,避免了使用支架材料带来的副作用。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (9)
1.一种牙髓干细胞膜片的制备方法,其特征在于,包括:
从牙髓组织中分离出干细胞;
将分离出的干细胞接种于细胞培养基中培养,其中,所述细胞培养基的配方为基础培养基添加转化生长因子β1和白介素10;
将培养后的干细胞接种于加入了基质金属蛋白酶9中和抗体的培养基;待处于所述培养基边缘处的干细胞出现皱褶时,将细胞膜片揭下。
2.根据权利要求1所述的制备方法,其特征在于,所述的基础培养基为α-MEM液体培养基。
3.根据权利要求1所述的方法,其特征在于,所述转化生长因子β1的浓度为30ng/ml-150ng/ml。
4.根据权利要求1所述的方法,其特征在于,所述白介素10的浓度为10ng/ml-100ng/ml。
5.根据权利要求1所述的方法,其特征在于,所述基质金属蛋白酶9中和抗体的浓度为1μg/ml-10μg/ml。
6.根据权利要求1所述的方法,其特征在于,制备成的细胞膜片包括1-6层牙髓干细胞DPSCs。
7.根据权利要求1所述的方法,其特征在于,所述从牙髓组织中分离出干细胞的步骤具体为:从健康的第三磨牙或前磨牙的牙髓组织中分离出干细胞。
8.根据权利要求1或2所述的方法,其特征在于,所述将培养后的干细胞制备成细胞膜片之前,还包括:
对所述培养后的干细胞进行鉴定。
9.根据权利要求8所述的方法,其特征在于,对所述培养后的干细胞进行鉴定的步骤包括:
检测所述培养后的干细胞是否能够进行骨向诱导分化,并
检测所述培养后的干细胞是否能够进行脂肪向诱导分化。
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