CN109234180B - 鞘氨醇单胞菌、其胞外产物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种鞘氨醇单胞菌、其胞外产物及其制备方法。所述鞘氨醇单胞菌(Sphingomonas sp.),其是保藏编号为CCTCC NO:M 2017211的鞘氨醇单胞菌JA‑MS‑001‑A‑04菌株。本发明还公开了鞘氨醇单胞菌的胞外产物在皮肤细胞增殖、修复及延缓衰老方面的应用,以及包含鞘氨醇单胞菌胞外产物的抗衰老、促进细胞增殖和/或迁移制剂。
Description
技术领域
本发明属于生物技术领域,具体涉及一种鞘氨醇单胞菌、其胞外产物及其胞外产物的制备,以及胞外产物在皮肤细胞增殖、修复及延缓衰老方面的应用。
背景技术
鞘氨醇单胞菌1990年才被科学家分离鉴定,直至2001年,其于分类学上因物理特性、系统发育等特点区别划分为4个属:鞘氨醇单胞菌属(Sphingomonas)、鞘氨醇菌属(Sphingobium)、新型鞘氨醇菌属(Novosphingobium)、鞘翅菌属(Sphigopyxis)。鞘氨醇单胞菌(Sphingomonas)是革兰氏阴性细菌,杆状、典型的好氧型、化学异养,具有独特的呼吸代谢途径-辅酶Q10,细胞膜组成中主要含有糖鞘脂而非脂多糖,通常分泌色素而使菌落呈黄色。鞘氨醇单胞菌属在环境微生物学领域受到越来越多的关注,其中,从污染环境中分离出来具有生物降解能力的菌株,可以降解多种化合物,尤其是芳烃类化合物。除此之外,鞘氨醇单胞菌属还具有生物合成能力,分泌胞外多聚物如结冷胶(Gellan Gum)、韦兰胶(Welan Gum)、鼠李糖胶(Rhamnose Gum)等,广泛应用于食品行业,主要依赖于多糖的化学特性及物理特性如粘性、凝胶性等和生物活性如抗肿瘤、抗氧化、降血脂血糖等。
皮肤组织从结构上由表皮层、真皮层、皮下组织及附属器官等构成。表皮层有大量角质形成细胞(keratinocytes,KC),表皮层最外一层是由5~20层已老化的角质细胞组成的角质层,具有对机械性刺激、物理、化学等因素的防护作用。正常情况下约30%的基底层细胞处于核分裂期新生的角质形成细胞有次序地逐渐向上移动由基底层移行至颗粒层约需14d,再移行至角质层表面并脱落又需14d,共约28d,称之皮肤细胞周期。角质形成细胞作为表皮重建的主要参与者,对皮肤的结构和功能起重要作用,与皮肤病变、创面愈合关系密切。胶原是真皮的主要成分,也是人体中含量最丰富的蛋白质,为皮肤提供强度和支撑。随着时间的流逝,这种皮肤的结构蛋白和主要组成成分逐渐退化,使皮肤呈现衰老的表现,Ⅰ型胶原是皮肤中含量最丰富、最重要的胶原类型。
近年来,有许多关于鞘氨醇单胞菌在皮肤上的应用,例如专利(US6,348,201)利用少动鞘氨醇单胞菌(Sphingomonas paucimobilis)IAM12576突变成白色菌株Sphingomonaspaucimobilis KFC-W-1,Sphingomonas paucimobilis MK-253W,Sphingomonaspaucimobilis MK-254W,Sphingomonas paucimobilis MK-332W等,经过培养,分离得到菌体,再通过甲醇、丙酮等具有挥发性的有机试剂提取菌体,得到含有鞘糖脂(sphingoglycolipid)的成分。该成分可以外用于皮肤,具有保湿或改善皮肤粗糙的功效。该提取方法限定了其活性成分来自于菌体,活性成分的效果较低,而且提取的过程中需要使用有机试剂,对工作人员和消费者都有一定的健康风险。
发明内容
因此本发明要解决的技术问题是:提供一种鞘氨醇单胞菌,其胞外产物能促进皮肤细胞增殖、迁移,以及延缓细胞的衰老。还提供了该鞘氨醇单胞菌的制备方法。
为解决上述技术问题,本发明采取的技术方案之一为:一种鞘氨醇单胞菌(Sphingomonas sp.),其是保藏编号为CCTCC NO:M 2017211的鞘氨醇单胞菌JA-MS-001-A-04菌株。
本发明采取的技术方案之一为:提供了一种本发明生产鞘氨醇单胞菌胞外产物的制备方法,其包括以下步骤:
(1)将鞘氨醇单胞菌接种入细菌常规培养基进行发酵培养;
(2)随后将发酵所得产物离心取上清液,即得;
较佳地,还可以包括:(3)将(2)所述上清液真空冷冻干燥,得干粉;
较佳地,发酵培养温度为30℃,时间为18~24h;离心速度为5000rpm,离心时间为10min。
更佳地,所述培养基含LB、GYT、SB、SOC、TB、2YT、胰蛋白大豆琼脂平板或牛肉膏蛋白胨培养基。
进一步更佳地,所述培养基包括蔗糖20g/L,酵母膏1g/L,K2HPO4 2g/L,MgSO4·7H2O 0.1g/L,调节pH 6.8~7.2。
本发明采取的技术方案之一为:本发明上述制备方法制得的鞘氨醇单胞菌胞外产物。
本发明采取的技术方案之一为:本发明上述鞘氨醇单胞菌胞外产物在促进细胞迁移、促进细胞增殖和/或延缓细胞衰老的制剂中的应用。较佳地,所述的细胞为人角质细胞。
本发明采取的技术方案之一为:包含本发明鞘氨醇单胞菌胞外产物的促进细胞增殖和/或促进细胞迁移的制剂。较佳地,所述的细胞为人角质细胞。
更佳地,胞外产物其终浓度为0.01-0.1%,所述百分比为质量百分比。
本发明采取的技术方案之一为:包含鞘氨醇单胞菌胞外产物的抗衰老制剂。较佳地,胞外产物的质量终浓度为0.001-0.1%;更佳地,所述胞外产物的终浓度为0.01-0.1%,所述百分比为质量百分比。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。如非说明书中特别指明,本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:本发明的鞘氨醇单胞菌产生的胞外产物具有良好的促进表皮细胞增殖、修复以及延缓细胞衰老的作用。
生物材料保藏信息
本发明的鞘脂单胞菌(Sphingomonas sp.)JA-MS-001-A-04,于2017年4月26日保藏在中国典型培养物保藏中心(CCTCC),地址为中国.武汉.武汉大学(邮编:430072),保藏编号为:CCTCC NO:M 2017211。
附图说明
图1为胞外产物对角质细胞增殖影响的柱状图,胞外产物的浓度分别为0.1%、0.01%和0.001%,*:p<0.05,**:p<0.01。
图2为胞外产物对角质细胞修复影响的柱状图,胞外产物的浓度分别为0.1%、0.01%和0.001%,*:p<0.05,**:p<0.01。
图3为胞外产物对角质细胞修复影响的组化图,其中A为胞外产物的效果;B为胞内裂解上清的效果。
图4为胞外产物对I型胶原合成影响的柱状图,胞外产物的浓度分别为0.1%、0.01%和0.001%,*:p<0.05,**:p<0.01。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1
鞘氨醇单胞菌的筛选和鉴定:从喜马拉雅地区的日多温泉水经过富集,用胰蛋白大豆琼脂平板(OXOID,酪蛋白胰酶水解物15.0g/L,大豆木瓜酶消化物5.0g/L,氯化钠5.0g/L,琼脂15.0g/L)分离,筛选疑似单菌落,然后进行2次划线菌种的纯化;纯化后的菌种,送至上海美吉生物科技公司进行菌种鉴定,使用细菌基因组DNA试剂盒(AxyPrep,细菌基因组DNA小量制备试剂盒AP-MN-BT-GDNA-250)将其DNA提取出来作为模板,进行PCR扩增。PCR使用的引物为16S通用引物27F(其序列为5'-AGA GTT TGA TCM TGG CTC AG-3')和1492R(其序列为5'-TAC GGY TAC CTT GTT ACG ACT T-3')。
PCR体系:
试剂 | 体积 |
10×Ex Taq buffer | 2.0μl |
2.5mM dNTP Mix | 1.6μl |
10mM 27F | 0.8μl |
10mM 1492R | 0.8μl |
DNA模板 | 0.5μl |
5u Ex Taq | 0.2μl |
ddH<sub>2</sub>O | 14.1μl |
Total volume | 20μl |
PCR反应条件:
电泳检测,切胶纯化测序,对其进行16SrDNA分子生物学分类鉴定,获得序列SEQID No.1,经Blast鉴定为鞘氨醇单胞菌(Sphingomonas sp.)种,命名为(Sphingomonassp.)JA-MS-001-A-04,于2017年4月26日保藏在中国典型培养物保藏中心(CCTCC),地址为中国.武汉.武汉大学(邮编:430072),保藏编号为:CCTCC NO:M 2017211。
实施例2
胞外产物制备:配置培养基(蔗糖20g/L,酵母膏1g/L,K2HPO4 2g/L,MgSO4·7H2O0.1g/L,调节pH约7.0)分装50ml于250ml锥形瓶,取实施例1筛选到的鞘脂单胞菌(Sphingomonas sp.)CCTCC NO.M 2017211 JA-MS-001-A-04菌株恒温震荡培养箱28~37℃(30℃左右最佳),约220rpm培养18~24h,菌种浓度OD600=0.6~0.8,接种比例1:100(体积比),发酵后5000rpm离心10~15min,移去菌体,收集上清,即为胞外产物粗提物,-20℃预冻过夜,真空冷冻干燥过夜,获得胞外产物粉末,冻存于-20℃。使用时,无菌去离子水配置成10mg/ml,用于后续实验。
上述发酵用的培养基,也可为细菌培养的常规培养基,如LB、GYT、SB、SOC、TB、2YT、胰蛋白大豆琼脂平板或牛肉膏蛋白胨培养基,这些培养基的区别仅在于配方中碳源、氮源及无机盐的含量稍有不同,因此将发酵条件如发酵时间略微进行调整即得。
胞内裂解上清的制备:移除的菌体用2~3倍体积PBS重悬,经过超声破碎(3s工作,3s暂停,共10min)超声2次,12,000rpm离心15min,除去细胞碎片,获得上清,冻存于-20℃,用于后续实验。
实施例3
角质细胞培养:人角质细胞(Keratinocyte,KC)使用完全培养基(Thermo)在37℃的5%CO2细胞培养箱培养,至90%铺板率时,胰蛋白酶消化后进行接种。
效果实施例1
KC增殖速度的测定:细胞以3,000/孔接种于96孔板,与不同浓度胞外产物溶液或胞内裂解上清(具体浓度见表1)37℃共培养1天后,移除完全培养基,加入MTT,3小时后小心吸去上清,加入二甲亚砜(DMSO),于550nm下进行检测;以未处理孔作为参照,定义样品孔为T,未处理孔为NT,以NT作为100%,样品孔T/NT的比值作为细胞增殖率。以NT作为1,T/NT>100%,说明该浓度物质对细胞增殖有促进作用,T/NT<100%,说明该浓度物质对细胞增殖可能存在毒性。图1及表1为胞外产物对角质细胞增殖的影响,胞外产物浓度在≤0.010%时,胞外产物对KC增殖有一定的促进作用。高浓度的裂解上清(浓度≥0.100%)会抑制KC的增殖,对细胞可能存在毒性。
表1.胞外产物和胞内裂解上清对角质细胞增殖的影响
效果实施例2
划痕实验:KC细胞以100,000个/孔接种于6孔板内,待细胞融合后用细胞刮在中央区域画一条线,线内的细胞被机械力去除,与不同浓度胞外产物溶液或胞内裂解上清(具体浓度见表2)37℃共培养1天后,吸去培养基,用PBS洗两次,冷甲醇固定,再用Giemsa染液对细胞进行染色,最后用流水冲洗,晾干。间隔一定时间通过观察细胞向无细胞的划痕区域迁移的情况,来判断细胞的迁移能力。以未处理孔作为参照,定义样品孔为T,未处理孔为NT,以NT作为100%,样品孔T/NT的比值作为细胞迁移率。以NT作为1,T/NT>100%,说明该浓度物质对细胞迁移有促进作用,T/NT<100%,说明该浓度物质对细胞迁移可能存在毒性。使用显微镜拍照监测划痕的闭合程度设置对照组和实验组,每个样品设置3次重复。计算每张图片的细胞覆盖面积和划痕面积比值,结果见表2、图2和图3A、3B,三个浓度均对细胞迁移有促进作用,有细胞的向无细胞区域迁移,0.010%的闭合程度最高,具有显著性差异。此外,胞内裂解上清对细胞迁移也有促进作用,能修复角质细胞的划痕。
表2.胞外产物和胞内裂解上清对角质细胞划痕面积的影响
效果实施例3
I型胶原含量测定:细胞以3,000/孔接种于96孔板与待测样品37℃共培养,24小时后,去上清,加入不含血清培养基(DMEM 100%,双抗P/S 1%)使胶原富集48小时,之后取孔板中上清,根据COL-I ELISA KIT说明书操作,进行人I型胶原含量的测定,于450nm下检测吸光值,以人I型胶原标准品(20~0ng/ml)系列做标准曲线。定义样品孔为T,未处理孔为NT。以未处理组作为对照组设置为100%,T/NT计算I型胶原蛋白增加值,结果参见图4及表3。>100%,表明该浓度物质能够促进I型胶原合成。由图4及表3可知,低浓度的胞外产物能很好的促进I型胶原的合成。此外,胞内裂解上清液能促进I型胶原的合成,但同浓度的裂解上清和胞外产物比,例如在0.100%浓度时,前者的促进作用稍逊于后者。
表3.胞外产物和胞内裂解上清对I型胶原合成的影响
图4及表3证实了该菌体培养液上清的冻干粉具有促进成纤维细胞I型胶原合成的能力。菌体胞内的裂解物上清其功效弱于培养液上清,在同等浓度即0.100%时,胞外产物显示出较高的功效,且胞外产物浓度低至0.001%,其促进胶原合成的能力高达193.22%。因此,鞘氨醇单胞菌的活性成分主要存在于胞外产物中,而非菌体内。该活性成分应不来自于或不完全来自于鞘糖脂,且该活性物的功效主要在于延缓皮肤衰老。
Claims (12)
1.一种鞘氨醇单胞菌(Sphingomonas sp.),其特征在于,其是保藏编号为CCTCC NO:M2017211的鞘氨醇单胞菌JA-MS-001-A-04菌株。
2.一种鞘氨醇单胞菌胞外产物的制备方法,其特征在于,其包括以下步骤:
(1)将如权利要求1所述的鞘氨醇单胞菌接种入细菌常规培养基进行发酵培养;
(2)随后将发酵所得产物离心取上清液,即得。
3.如权利要求2所述的胞外产物的制备方法,其特征在于,所述制备方法还包括:(3)将(2)所述上清液真空冷冻干燥,得干粉。
4.如权利要求2或3所述的胞外产物的制备方法,其特征在于,所述常规培养基为LB、GYT、SB、SOC、TB、2YT、胰蛋白大豆琼脂平板或牛肉膏蛋白胨培养基。
5.如权利要求4所述的胞外产物的制备方法,其特征在于,所述常规培养基含蔗糖20g/L,酵母膏1g/L,K2HPO4 2g/L,MgSO4·7H2O 0.1g/L,调节pH 6.8~7.2。
6.如权利要求2所述的制备方法,其特征在于,所述的发酵培养的温度为30℃,时间为18~24h;和/或所述离心速度为5000rpm,离心时间为10min。
7.如权利要求2~6任一项所述的制备方法制得的鞘氨醇单胞菌胞外产物。
8.如权利要求7所述的鞘氨醇单胞菌胞外产物在制备促进细胞迁移、促进细胞增殖和/或延缓细胞衰老的制剂中的应用;其特征在于,所述鞘氨醇单胞菌胞外产物的终浓度为0.001-0.1%,所述百分比为质量百分比;
所述的细胞为人角质细胞。
9.一种包含权利要求7所述的鞘氨醇单胞菌胞外产物的促进细胞增殖和/或促进细胞迁移的制剂;其特征在于,所述鞘氨醇单胞菌胞外产物的终浓度为0.001-0.1%,所述百分比为质量百分比;
所述的细胞为人角质细胞。
10.如权利要求9所述的制剂,其特征在于,所述鞘氨醇单胞菌胞外产物其终浓度为0.01-0.1%,所述百分比为质量百分比。
11.一种包含权利要求7所述的鞘氨醇单胞菌胞外产物的抗衰老制剂;其特征在于,所述胞外产物的质量终浓度为0.001-0.1%,所述百分比为质量百分比。
12.如权利要求11所述的制剂,其特征在于,所述胞外产物的终浓度为0.01-0.1%,所述百分比为质量百分比。
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