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1、<p><p>&lt;p&gt;高雄醫學大學口腔醫學院 研究日 第五屆學術研討會,26 April, 2013,Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation,Yuk-Kwan Chen1,3 Anderson Hsien-Cheng Huang1 Tien-Yu S
2、hieh2,4 Li-Min Lin1,3 Departments of 1Oral Pathology, 2Oral Division of Dentistry, 3Departments of Oral Pathology, 4Oral &amp;amp; Maxillofacial Surgery, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan,Overall Introduction,Adult stem cells (ASCs) have been isolated
3、 from various tissues including bone marrow, dental, liver, blood vessel, adipose, and neuronal tissues (Barrilleaux et al., 2007) Stem cells isolated from dental epithelium and dental pulp are capable of differentiating to a dentin-pulp-like complex, which is similar to that of normal human teeth (
4、Gronthos et al., 2000; Gronthos et al., 2002),Human dental pulp stem cells (hDPSCs) share similar gene expression profiles and differentiation capacity to that of bone marrow mesenchymal stem cells (BMSCs) (Shi et al., 2001) hDPSCs are also capable of differentiating to osteogenic, adipogenic, chond
5、rogenic, and neurogenic lineages (Gronthos et al., 2000; Gronthos et al., 2002; Iohara et al., 2006), but not for hepatic differentiation of cryopreserved dental pulp tissues of vital extracted teeth with disease,Overall Introduction,Part (1) Different Cryopreservations of Human Dental Pulp Tissues
6、from Vital Extracted Teeth with Disease Part (2) Hepatic-Like Differentiation from Cryopreserved Dental Pulp Tissues of Vital Extracted Teeth with Disease,The current study contains the following two parts of experiments, Different Cryopreservations of Human Dental Pulp Tissues from Diseased Teeth,G
7、roup A Freshly derived dental pulp tissues, n = 20 Age: range - 6-74 y/o average - 26.5 y/o Sex: M/F: 8/12,Group B Liquid nitrogen (N2) stored dental pulp tissues, n = 20 Age: range - 6-49 y/o average - 23.4 y/o Sex: M/F: 5/15,Group C Liquid nitrogen (N2) stored intact teeth, n =10 Age : range - 8-5
8、2y/o average - 27.7y/o Sex: M/F: 5/5,Total sample size, n = 50 Age: range - 6-74 y/o average - 25.5 y/o Sex: M/F: 18/32,M/F: Male/Female,A cohort of 50 vital extracted teeth with diseases (residual root, supernumerary tooth, periodontitis, pericoronitis, and tooth fracture) were randomly divided as:
9、 Group A (n = 20) : Freshly derived dental pulp tissues Group B (n = 20) : Liquid nitrogen stored dental pulp tissues for 30 days (dump-freezing process at -1&amp;#176;C/min for over 8-h in -80&amp;#176;C after immersing in 1ml culture medium with 10% DMSO for 2h at 4&amp;#17
10、6;C) Group C (n = 10) : Liquid nitrogen stored intact teeth for 30 days (dump-freezing process at -1&amp;#176;C/min for over 8-h in -80&amp;#176;C after immersing in 1ml culture medium with 10% DMSO for 2h at 4&amp;#176;C),Materials,Growth rate analyses by MANOVA on sex, age
11、&amp;amp; tooth position categories for Group A samples,The whole model result of MANOVA on Group A (n = 20),The whole model result of MANOVA on Group B samples (n = 20),Growth rate analyses by MANOVA on sex, age &amp;amp; tooth position categories for Group B samples,A circumference
12、 groove of 0.5-1.0 mm in depth was cut around the entire tooth (arrows) using an aseptic high speed handpiece bur after cleaning the tooth surface with DPBS (A). The tooth was split using a chisel (B), dental pulp was then exposed (C) and the underlying pulp tissue was extracted using an endodontic
13、file placing in a container with DPBS for subsequent procedures of culture (D),Cryopreservation vial containing the intact tooth,Group A Group B,Group C,Isolation and culture of human dental pulp stem cells (DPSCs ) Colony forming unit (CFU),Methods and Results,Growth rate analyses,The hDPSCs of Gro
14、ups AC had a high proliferation rate during the early passages, but decreased gradually in culture.,Osteogenic, adipogenic, and chondrogenic differentiation,Group B samples (successful isolation, n = 20),Group C samples (successful isolation, n = 2),Reverse transcription-polymerase chain reaction (R
15、T-PCR),GAPDH (596-bp) Osteonectin (323-bp) Nestin (810-bp) Nanog (389-bp) Rex-1 (470-bp) Oct-4 (205-bp),RT-PCR for stem cell (Oct-4, Nanog &amp;amp; Rex-1) and differentiation (Osteonectin, Nestin and PPAR) markers of Group A samples (successful isolation, n = 20),RT-PCR for stem cell (Oct-4
16、, Nanog and Rex-1) and differentiation (Osteonectin, Nestin and PPAR) markers of Group B samples (successful isolation, n = 20),RT-PCR for stem cell (Oct-4, Nanog &amp;amp; Rex-1) and differentiation (Osteonectin, Nestin and PPAR) markers of Group C samples (successful isolation, n = 2),GAPD
17、H (596-bp) Osteonectin (323-bp) Nestin (810-bp) Nanog (389-bp) Rex 1 (470-bp) Oct4 (205-bp),H2O,Flow cytometry,Flow cytometry of the isolated hDPSCs of Group B samples (successful isolation, n = 20),CD29, CD34, CD45, CD90, CD105 are important markers for human bone marrow stem cells which show similar expression to our isolated hPDSCs (Black line: markers of isotype; Red line: markers of interes&lt;/p&gt;</p></p>
