An Kangkang, Liu Hongwei
Department of Periodontology, Laboratory of Oral Biomedical Science and Translational Medicine, School of Stomatology, Tongji University, Shanghai 200072, China.
Department of Periodontology, Laboratory of Oral Biomedical Science and Translational Medicine, School of Stomatology, Tongji University, Shanghai 200072, China. Email:
Zhonghua Kou Qiang Yi Xue Za Zhi. 2014 Nov;49(11):682-7.
To evaluate the survival of bone marrow mesenchymal stem cells (BMMSC) and periodontal ligament stem cells (PDLSC) in BMMSC/PDLSC cell sheets which transplanted ectopically into subcutaneous dorsum of nude mice.
The canine BMMSC and PDLSC from primary culture were tranfected with lentiviral vectors carrying green fluorescent protein (GFP) gene (Lentivirus-GFP) or red fluorescent protein (RFP) gene (Lentivirus-RFP) respectively. The immunophenotypes of GFP-labeled BMMSC and RFP-labeled PDLSC were identified by flow cytometry. Adipogenic and osteogenic differentiation of them were detected by alizarin red or oil red O respectively. Then, both GFP-labeled BMMSC cell sheets and RFP-labeled PDLSC cell sheets were fabricated respectively using normal culture dish (6 cm) after stimulation of extracellular matrix formation. Each was enveloped by collagen membrane (Bio-Gide) and then transplanted into the subcutaneous dorsum of nude mice. In vivo non-invasive biofluorescence imaging(BFI) was performed at 1, 2, 4 and 8 w post-tranplantation to trace and quantify the survival and growth of RFP-labeled PDLSC and GFP-labeled BMMSC via the BFI system of the NightOWL. The fluorescence intensity change of GFP/RFP signal was monitored and compared. The mice were sacrificed 8 weeks after cell sheets transplantation and the survival of stem cells was verified by fluorescence immunohistochemistry.
The flow cytometry showed that GFP-labeled BMMSC positively expressed CD29, CD44, CD34, STRO-1 were 93.07%, 92.84%, 3.23%, 67.67%, and RFP-labeled PDLSCs were 89.91%, 88.47%, 6.04%, 74.11%, respectively. Both of them had the potency of differentiating into osteoblasts and adipocytes. The stemness of both of them was almost same. After being transplanted into nude mice, the signal strength of GFP(BMMSC) was weaker and weaker in 1, 2, 4 and 8 w [(83.1±3.1)×10(6), (65.1±2.3)×10(6), (51.5 ± 2.3)×10(6), (33.8 ± 2.0)×10(6) ph/s, respectively.]. The signal strength of RFP(PDLSC) was weakenedin 1, 2 and 4 w [(53.8±3.0)×10(6), (42.2±2.6)×10(6), (34.5±2.1)×10(6) ph/s], then recovered in 8 w ([ 45.1±2.9)×10(6) ph/s]. The signal strength of RFP(PDLSC) was signifcantly stronger in 8 w than in 4 w(P < 0.01). The survival of RFP-labeled PDLSC was significant higher than that of GFP-labeled BMMSC. After 8 weeks, lots of RFP-labeled PDLSC were observed by microscope, but less GFP-labeled BMMSC were observed.
Histometric analysis revealed that the survival of stem cells in the RFP-labeled PDLSC cell sheets was significantly higher than that of in the GFP-labeled BMMSCs cell sheets.
评估异位移植到裸鼠背部皮下的骨髓间充质干细胞（BMMSC）/牙周膜干细胞（PDLSC）细胞片中BMMSC和PDLSC的存活情况。
将原代培养的犬BMMSC和PDLSC分别用携带绿色荧光蛋白（GFP）基因的慢病毒载体（慢病毒-GFP）或红色荧光蛋白（RFP）基因的慢病毒载体（慢病毒-RFP）转染。通过流式细胞术鉴定GFP标记的BMMSC和RFP标记的PDLSC的免疫表型。分别用茜素红或油红O检测它们的成脂和成骨分化。然后，在刺激细胞外基质形成后，分别使用普通培养皿（6 cm）制备GFP标记的BMMSC细胞片和RFP标记的PDLSC细胞片。每片均用胶原膜（Bio-Gide）包裹，然后移植到裸鼠背部皮下。在移植后1、2、4和8周进行体内无创生物荧光成像（BFI），通过NightOWL的BFI系统追踪和量化RFP标记的PDLSC和GFP标记的BMMSC的存活和生长情况。监测并比较GFP/RFP信号的荧光强度变化。细胞片移植8周后处死小鼠，通过荧光免疫组织化学验证干细胞的存活情况。
流式细胞术显示，GFP标记的BMMSC中CD29、CD44、CD34、STRO-1的阳性表达率分别为93.07%、92.84%、3.23%、67.67%，RFP标记的PDLSC中分别为89.91%、88.47%、6.04%、74.11%。它们均具有分化为成骨细胞和脂肪细胞的能力。两者的干性几乎相同。移植到裸鼠体内后，GFP（BMMSC）在1、2、4和8周时的信号强度逐渐减弱[分别为(83.1±3.1)×10(6)、(65.1±2.3)×10(6)、(51.5 ± 2.3)×10(6)、(33.8 ± 2.0)×10(6) ph/s]。RFP（PDLSC）在1、2和4周时信号强度减弱[(53.8±3.0)×10(6)、(42.2±2.6)×10(6)、(34.5±2.1)×10(6) ph/s]，然后在8周时恢复[ (45.1±2.9)×10(6) ph/s]。RFP（PDLSC）在8周时的信号强度显著强于4周时（P < 0.01）。RFP标记的PDLSC的存活率显著高于GFP标记的BMMSC。8周后，显微镜下观察到大量RFP标记的PDLSC，但观察到的GFP标记的BMMSC较少。
组织计量学分析显示，RFP标记的PDLSC细胞片中干细胞的存活率显著高于GFP标记的BMMSC细胞片。
