两种人包皮成纤维细胞分离培养方法的比较

Abstract

Objective

The efficient acquisition and purification of fibroblasts as ideal seed cells are very important. For optimization of the isolation and culture of human foreskin fibroblasts (HFF), we compared the improved tissue culture method (ITCM) and the enzyme digestion method (EDM).

Methods

In ITCM, the skin tissue was digested with 0.1% Type II collagenase overnight at 4 ℃, the epidermis was separated from the dermis and digested again with 0.25% trypsin at room temperature for 15 min, and then the tissue block was attached to the culture dish. In EDM, the skin tissue was digested with 0.25% trypsin overnight at 4 ℃, the epidermis was separated from the dermis and digested with 0.1% Type II collagenase overnight at 4 ℃, the tissue block was filtered and squeezed together with the enzyme mixture, the filter was rinsed with medium containing fetal bovine serum, and the cell suspension was cultured. Both ITCM and EDM used 2 digestion enzymes, but the order, digestion time, and temperature of the 2 enzymes were different. The final inoculations of ITCM and EDM in the dishes for subsequent culture were tissue blocks and cell suspensions, respectively. In this study, HFF cells were isolated and cultured with ITCM and EDM, and the cell morphology was observed from Passage 0 to Passage 3 in the ITCM and EDM groups. The cell purity was identified by staining for vimentin, CD68, and Pan-keratin. The growth curves of Passage 3 were plotted to compare the proliferation ability of the 2 groups. Passage 3 HFF cells in the ITCM and EDM groups were irradiated with medium-wave ultraviolet (UVB) at an energy value of 120 mJ/cm² to establish a light damage model. The experiments were grouped into an UVB group and a control group (Control) according to the presence or absence of UVB irradiation. Platelet-poor plasma (PPP) was extracted by secondary centrifugation, and the HFF cells of ITCM and EDM groups were cultured in groups using complete medium containing different concentrations (0, 2.5%, 5.0%, and 10.0%) of PPP, and the proliferation of damaged cells was detected by cell counting kit-8 after 24 h of PPP incubation.

Results

A large number of HFF could be observed in the ITCM group up to day 3, which was less affected by impurities; the observation of HFF morphology in the EDM group was affected by more impurities. By day 9, cells in both ITCM and EDM groups could be passaged; HFF isolated and cultured in vitro by the 2 methods showed long spindle-shaped, swirling growth. The positive rates of vimentin in the ITCM and EDM groups when HFF cells were cultured up to Passage 2 were significantly different [(97.36±0.76)% vs (99.4±0.56)%, P<0.01)]. The positive rates of CD68 were also significantly different [(70.8±0.46)% vs (78.37±0.75)%, P<0.01]. The expressions of pan-keratin in the ITCM group and the EDM group were positive and negative, respectively. There was no difference in vimentin and pan-keratin staining results between the ITCM group and the EDM group when HFF were cultured to Passage 3. The positive rates of CD68 between the ITCM group and the EDM group were significantly different [(74.73±1.37)% vs (85.27±2.63)%, P<0.001]. The proliferative capacity of HFF cells in Passage 3 was significantly higher in the EDM group than that in the ITCM group (P<0.05). After UVB (120 mJ/cm²) irradiation, HFFs procured by the 2 isolation methods showed damage. The damage repair test demonstrated that the 2.5% PPP+UVB irradiation group showed significantly higher repair competence than the other groups (all P<0.05).

Conclusion

In contrast with HFFs isolated via ITCM, HFF cells isolated by EDM have a faster purification rate and a stronger proliferative capacity. Therapy with PPP can moderately repair UVB-induced damage to HFFs. The results provide a theoretical basis for clinical treatment studies in the future.

成纤维细胞是皮肤研究领域的典型细胞模型细胞之一^[1-4]。传统的成纤维细胞分离培养方法包括组织块培养法(tissue culture method，TCM)及酶消化法(enzyme digestion method，EDM)^[5]。近年来，已有大量的学者^[6-9]分别利用两者之一提取人皮肤成纤维细胞，应用于种子细胞获取、组织细胞工程、皮肤伤口愈合和信号转导机制等方面的研究；亦有学者^{[5, 10]}比较两种方法的优劣，但是对于哪种方法更佳仍有争议。本研究采用改良组织块培养法(improved tissue culture method，ITCM)及EDM对人包皮成纤维细胞(human foreskin fibroblast，HFF)进行分离培养，在显微镜下观察细胞形态，采用细胞免疫荧光对原代至第3代(Passage 0~Passage 3，P0~P3)细胞进行鉴定，绘制P3细胞的生长曲线；建立UVB光损伤模型并进行贫血小板血浆(platelet-poor plasma，PPP)治疗，以评价两种方法的优劣，为后续临床治疗研究提供理论依据。

1. 材料与方法

1.1. 主要试剂及仪器

DMEM、II型胶原酶、胰酶、胎牛血清(fetal bovine serum，FBS)和青霉素-链霉素双抗购自美国Gibco公司；细胞过滤筛购自美国BD公司；细胞计数试剂盒-8(cell counting kit-8，CCK-8)购自日本同仁化学研究所；4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole，DAPI)染色液、波形蛋白(vimentin)标记抗体、CD68兔单克隆抗体、角蛋白(pan-keratin)标记抗体及二抗均购自美国CST公司。UVB灯管TL 20W/01购自德国Philips公司；CO₂恒温细胞孵育箱购自美国Thermo公司；低速离心机KDC-40购自安徽中科中佳科学仪器有限公司；研究级倒置相差荧光显微镜IX71购自日本Olympus公司；酶标仪X-Mark购自美国Bio-Rad公司。

1.2. 方法

1.2.1. PPP的制备

抽取健康成人志愿者全血约20 mL，以1 500 g离心10 min, 取交界面以下1 mm处的上清液，再次以200 g离心10 min，管内总液体的上3/4即为PPP，将之分装并置于-20 ℃冰箱备用。

1.2.2. HFF的分离和培养

本项目获得海口市人民医院医学伦理委员会批准(批准号：SC20170122)。收集无菌包皮环切术标本，置于无菌低温条件下，立即送往实验室。使用含10%青霉素-链霉素双抗的PBS冲洗包皮组织5次，剔除皮下组织。将剩余皮肤组织剪成约3 mm×3 mm小块，用PBS冲洗2次，平均分为2份备用。

1.2.2.1. ITCM

取一份样本，加入0.1% II型胶原酶在4 ℃下培养过夜。第2天吸去胶原酶，分离表皮及真皮。在真皮样本中加入0.25%胰酶于室温下消化15 min后，用完全DMEM终止。混合液以800 r/min离心8 min后弃上清，然后将组织块接种于培养皿中，彼此间隔3~5 mm，再加入完全培养基，置于37 ℃，5% CO₂培养箱中培养，每3 d换液1次。

1.2.2.2. EDM

取备用的另一份样本，加入0.25%胰酶于4 ℃下培养过夜。第2天吸去胰酶，分离并丢弃表皮，加入0.1% II型胶原酶于4 ℃下继续培养过夜。然后将组织块及胶原酶一起过滤，反复多次按压细胞筛上的组织块，同时加入含有10% FBS的DMEM冲洗细胞筛。将得到的细胞悬液接种于培养皿中，置于37 ℃，5% CO₂培养箱中培养。第2天换液，之后每3 d换液1次。

1.2.3. 细胞传代

待细胞长至80%~90%融合时，弃培养基，以PBS清洗2遍，加入0.25%EDTA-胰酶于37 ℃下消化2 min，然后使用完全培养基终止消化。按照1꞉3进行细胞传代。待细胞传至P3，进行后续实验。

1.2.4. 细胞鉴定

将P0~P3 HFF接种于6孔板直至检测时间。吸去6孔板中的细胞培养基，以PBS洗3遍。每孔加入甲醇或4%多聚甲醛溶液1 mL；于室温下固定15 min，弃固定液并清洗，加入1 mL 0.1% Triton X-100 于室温下孵育 15 min。清洗后加入1 mL 1% BSA 于室温下封闭45 min。弃上清液，分别加入1 mL波形蛋白兔单克隆抗体(1꞉100)、CD68兔单克隆抗体(1꞉800)、角蛋白鼠单克隆抗体(1꞉400)，于4 ℃下孵育过夜。第2天取出6孔板恢复至室温，清洗后每孔分别加入 1 mL对应的荧光二抗，于室温下孵育1 h。清洗后加入0.2 mg/L DAPI于室温下避光孵育5 min。再次清洗后在倒置相差荧光显微镜下观察拍照。实验中的清洗均为PBS洗3遍，每遍5 min。在两种方法获取的P2及P3 HFF中，计数波形蛋白和CD68阳性细胞，用DAPI染核以计数总细胞，算出波形蛋白和CD68阳性率。计算公式为：阳性率=(阳性细胞数/总细胞数)×100%。

1.2.5. 绘制生长曲线

将两种方法获得的P3 HFF接种于96孔板，接种密度为1×10⁴个细胞/mL，每孔接种100 µL，共接种7板，置于37 ℃、5% CO₂培养箱中；分别在第1，2，3，4，5，6，7天，每孔加入10 µL CCK-8试剂，培养2 h后测量450 nm波长处的光密度(optical density，OD)值，用于绘制生长曲线。

1.2.6. UVB光损伤模型的建立及PPP治疗

将P3 HFF接种于96孔板，每孔4×10³个细胞，24 h后开始照射UVB(UVB组)或置于关闭的UVB灯管下(对照组)。照射前弃去上清液，每孔加入PBS 50 µL；照射后更换为含有1% FBS的DMEM。每12 h照射1次，连续照射4次。最后1次照射后，更换为含有不同浓度(0、2.5%、5%和10%)PPP的DMEM进行修复培养。培养24 h后分别加入CCK-8试剂对细胞增殖情况进行检测。

1.3. 统计学处理

采用SPSS 20.0统计学软件进行数据分析，实验数据采用均数±标准差( $\overline{x}$ ±s)表示。两组数据的比较采用独立样本t检验，多组数据的比较采用单因素方差分析(one-way ANOVA)。P<0.05为差异具有统计学意义。

2. 结 果

2.1. 两种方法分离培养得到的HFF形态

图1所示两种方法获得的HFF形态。图1A显示：第3天，ITCM可见组织块边缘有大量的成纤维细胞，EDM偶见成纤维细胞且杂质较多；第6天，两组细胞明显增多，细胞呈长梭形、多角形；第9天时细胞可传代。图1B显示：P0~P1 ITCM组以大的杂细胞团为主，EDM组中的杂细胞由数量多而密集变为体积小且分散。两组细胞P2均有散在的较圆的杂细胞。当细胞传代至P3时，两种方法获得的HFF均呈长梭形、旋涡状生长，两组细胞形态一致。

图1.

两种方法获得的HFF形态

Figure 1 Morphology of HFF isolated from the 2 methods

A: HFF morphology of ITCM and EDM on the 3rd, 6th, and 9th day (×100); B: HFF morphology of ITCM and EDM from P0 to P3 (×100). ITCM: Improved tissue culture method; EDM: Enzyme digestion method; HFF: Human foreskin fibroblast.

2.2. 细胞鉴定

真皮成纤维细胞的波形蛋白、CD68表达阳性，细胞呈绿色荧光；上皮角质细胞角蛋白表达阳性，细胞呈绿色荧光。如图2所示，两种方法获取的HFF 细胞：在P0，ITCM组的细胞含有较集中的杂细胞，EDM组的杂细胞则较为分散；在P1，ITCM组的细胞仍有较大、较集中的杂细胞团，EDM组的杂细胞则更加分散、细小；细胞传至P2时，用ITCM获取的细胞中仍然可见杂细胞，波形蛋白及CD68阳性率[分别为(97.36±0.76)%和(70.8±0.46)%]显著低于EDM获取的波形蛋白及CD68的阳性率[分别为(99.4±0.56)%和(78.37±0.75)%]，角蛋白的表达为ITCM阳性、EDM阴性；P3时两组细胞均无杂细胞，波形蛋白染色阳性率均达到100%，CD68染色结果显示ITCM的阳性率[(74.73±1.37)%]显著低于EDM的阳性率[(85.27±2.63)%]，两组细胞角蛋白染色结果均为阴性。综上，EDM在短时间内获得纯化细胞的能力最佳。

图2.

P0~P3 HFF鉴定

Figure 2 Identification of HFF in P0-P3

Vimentin (A), CD68 (B), and pan-keratin (C) expressions in P0-P3 HFF isolated by the 2 methods. Green fluorescence is positive expression and blue fluorescence is DAPI-stained (×100). Positive rate of vimentin and CD68 (D) by the ITCM and EDM. **P<0.01, ***P<0.001. ITCM: Improved tissue culture method; EDM: Enzyme digestion method; HFF: Human foreskin fibroblast; BF: Bright field; DAPI: 4',6-diamidino-2-phenylindole.

2.3. 生长曲线绘制

两种方法获取的P3HFF细胞，在接种后第0~1天，细胞处于潜伏期；第2~3天，细胞处于增殖期；第4~5天，细胞数量明显增加，处于对数生长期；第6~7天，细胞数量增加减少，进入平台期。通过EDM获得的HFF细胞在第2~5天，细胞的增殖能力显著高于ITCM获取的细胞(图3)。

图3.

P3 HFF增殖曲线

Figure 3 Passage 3 of HFF growth curve

*P<0.05, **P<0.01, ***P<0.001 vs ITCM at the same check point. ITCM: Improved tissue culture method; EDM: Enzyme digestion method; HFF: Human foreskin fibroblast.

2.4. 建立UVB光损伤模型

经过4次UVB照射后，用ITCM及EDM获取的细胞数量及形态均发生明显变化。与未照射的对照组比较，细胞数量明显减少，细胞形态由长梭形变为胞体增宽、增大的扁平状，细胞质及细胞核变宽并有颗粒状物质(图4)。

图4.

UVB光照后HFF细胞形态(×100)

Figure 4 Morphology of HFF after UVB irradiation (×100) ITCM: Improved tissue culture method; EDM: Enzyme digestion method; HFF: Human foreskin fibroblast; UVB: Ultravoiolet B.

2.5. 不同浓度的PPP对P3 HFF光损伤修复的影响

加入含PPP培养基培养后，对照组HFF形态均为典型的长梭状；UVB组细胞形态随PPP的浓度不同有所差异(图5A)。两种方法在对照组组内，与0 PPP组相比较，其余各组细胞增殖数量明显增加；在UVB组组内，2.5% PPP治疗组的细胞增殖优于其余各组(P<0.05或P<0.001，图5B)。在对照组细胞之间比较，EDM获取的HFF细胞增殖能力明显高于ITCM。

图5.

PPP治疗后HFF细胞形态及增殖变化

Figure 5 Damage repair of HFF cells after PPP treatment

A: Morphology of cells after PPP treatment (×100); B: Proliferation of cells after PPP treatment. *P<0.05, ***P<0.001 vs control+0 PPP; †P<0.05 vs UVB+0 PPP, UVB+5.0% PPP, and UVB+10.0% PPP groups, †††P<0.001 vs the UVB+0 PPP, UVB+5.0% PPP, and UVB+10.0% PPP groups; ‡‡‡P<0.001 vs the Control+2.5% PPP, Control+5.0% PPP, and Control+10.0% PPP groups. ITCM: Improved tissue culture method; EDM: Enzyme digestion method; UVB: Ultravoiolet B; PPP: Platelet-poor plasma, HFF: Human foreskin fibroblasts.

3. 讨 论

传统的组织块培养法是将皮肤组织直接剪碎后置于培养皿中^[6]；或者利用一种酶的消化作用分离皮肤组织的表皮及真皮^[7]，将真皮组织剪碎后进行培养。本实验对组织块培养法进行改良，组织块共经过两种酶依次消化，首次酶消化后将表皮组织分离并丢弃，对真皮组织再次酶消化后加入少量培养基进行接种培养。第3天换液时可见大量的成纤维细胞从组织块边缘爬出，培养至第9天，当细胞融合度达到80%时进行传代，之后按照1꞉3每3天传代1次。以往采用传统组织块培养法，细胞爬出组织块时间为最快4~6 d^[11]，最慢1~2周^[6-7]；细胞首次传代时间为21~24 d^[10]，再次传代时间为3~6 d^[11]、7~8 d^{[6, 10]}。本研究中ITCM在培养效率方面与传统的方法相比具有更大的优势。EDM是将皮肤组织块用一种酶消化^[8]或者两种酶依次消化后，直接分离成纤维细胞进行培养^[12]。本实验采用两种酶依次消化的方法获取细胞悬液。结果显示：ITCM与EDM比较，前者细胞纯化所需时间更久，增殖能力更弱；后者具有增殖能力强、利于纯化的特点。

紫外线导致皮肤光损伤并能诱发皮肤恶性肿瘤，其作用光谱中UVB(290~320 nm)虽然波长较短，但能穿透表皮到达真皮，进而影响成纤维细胞产生强大的生物学效应^[13-15]。Zeng等^[14]将P1~P3小鼠真皮成纤维细胞反复暴露于4种产生亚细胞毒性剂量的UVB下，证实120 mJ/cm² UVB照射能够成功构建稳定的细胞衰老模型。PPP是提取富血小板血浆后残留的血浆，含有有益的蛋白质、胰岛素和少量的血小板^[16]。目前富血小板血浆作为成纤维细胞的增殖剂已被大量研究，然而PPP的相关研究较少^[17]。本次实验采用120 mJ/cm² UVB 4次照射导致P3 HFF出现光损伤形态，经PPP治疗后细胞损伤逐渐被修复。实验结果进一步验证了两种方法获取的HFF细胞均可用于构建光损伤模型以及PPP治疗的可行性。

成纤维细胞是起源于中胚层的间充质细胞，具有典型的细长纺锤形形态，通过标记间充质来源的阳性抗体及其他谱系(例如上皮和造血)的阴性抗体进行鉴别^[18]。波形蛋白为III型中间丝蛋白，主要表达于间充质细胞中，被认为是成纤维细胞鉴定实验中的阳性抗体，波形蛋白阳性率接近100%的成纤维细胞被视为纯化良好^{[5-6, 11, 19]}。角蛋白是上皮细胞分化的最常见标志物，在成纤维细胞鉴定实验中被用来作为阴性抗体使用^[20]。王志强等^[10]的实验佐证了P4成纤维细胞的纯度。CD68是一种高度糖基化的糖蛋白，作为重要的标志物应用于单核/巨噬细胞、肿瘤细胞、成纤维细胞和内皮细胞在炎症组织、肿瘤组织以及其他免疫组织的病理学诊断中^[21-23]。本研究采用上述3种抗体对两种方法获取的P0~P3 HFF进行免疫荧光染色，计算P2~P3细胞波形蛋白和CD68的阳性率，采用定性的方式描述角蛋白的表达。结果显示：随传代次数增加波形蛋白阳性率越来越高；CD68的阳性率虽然未达到100%，但其阳性率的变化趋势与波形蛋白一致，同样随传代次数的增加而增加。这提示CD68可能起到辅助鉴定的作用。而角蛋白的表达随传代次数增加逐渐消失。P0~P1 HFF受到成团、分层生长的杂细胞影响，无法计算阳性及总细胞数量，未计算其阳性率。因此，在成纤维细胞鉴定实验中，以波形蛋白为首选标志物的基础上，增加对角蛋白及CD68的检测可以更好地鉴定成纤维细胞及判断其纯度，二者可以作为鉴定成纤维细胞的辅助标志物。

综上，采用EDM分离培养HFF具有利于纯化、增殖高效的优势，ITCM较传统组织块培养法具有培养效率高的特点，两种分离培养方法均可用于构建光损伤及治疗模型。

基金资助

海南省自然科学基金(818QN325)；海南省卫生健康行业科研项目(20A200475)。

This work was supported by the Natural Science Foundation of Hainan Province (818QN325) and the Health Profession Research Project of Hainan Province (20A200475), China.

利益冲突声明

作者声称无任何利益冲突。

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202108800.pdf