2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 171 DOI:10.3969/cmba.j.issn.1673-713X.2009.03.003 BALB/c 2 3 7 14 HE -1 OP-1 G-CSF 3 6 9 12 15 12 CD34-1 Sca-1 12 OP-1 G-CSF MTT HE OP-1 9 12 15 G-CSF 9 P < 0.05 OP-1 CD34 Sca-1 13.07% ± 0.19% 3.98% ± 0.15% 17.02% ± 0.12% 11.62% ± 0.44% 3.03% ± 0.11% 2.91% ± 0.14% P < 0.05 G-CSF CD34 Sca-1 12.79% ± 0.39% 4.52% ± 0.35% 10.21% ± 0.15% P < 0.05 MTT OP-1 G-CSF P < 0.05 OP-1 www.cmbp.net.cn, 2009, 4(3):171-176 [1] [2] muscle-derived stem cells MDSC [3-5] [6] [7] [8] CD34 CD45-1 stem cell antigen-1 Sca-1 glial fibrillary acidic protein GFAP alphafetoprotein AFP CD105 [9] -1 osteogenic protein-1 OP-1 -β TGF-β [10] G-CSF OP-1 G-CSF 30571766 Y2008C06 250062 Email shili2005@sina.com 2009-01-05
172 2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 DMEM Gibco Hyclone PE CD34 FITC Sca-1 Ly6A.2/6E.1 BioLegend OP-1 071206 G-CSF 080102 MTT 2 mm 20 mm 50 mm absorbable gelatin sponge AGS 90% 2 mm 20 mm 50 mm polymer of lactic acid PLA 90% FACS Calibur BD KD-TS3C KD-BM KD-H HM315 MICROM Synergy HT Bio-TEK ALC ACCULAB SPF BALB/c 5 ~ 6 12 ~ 15 g SCXK 20030004 SPF 0.25 mg/ml OP-1 24 ~ 48 h 1.6 mg/ 5 mm 2 mm BALB/c 12 6 2 3 7 14 2 HE OP-1 G-CSF BALB/c 30 OP-1 G-CSF 10 OP-1 OP-1 0.25 mg/ml G-CSF 250 µg kg -1 d -1[11] 7 d G-CSF 3 6 9 12 15 2 D-Hanks DMEM 100 3 12 1 10 6 /ml PBS 3 PE CD34 FITC Sca-1 37 30 min PBS 2 CD34 Sca-1 3 OP-1 G-CSF 12 10% 10% DMEM 5 10 4 /ml 100 µl/ 96 3 3 d 50 ng/ml OP-1 OP-1 50 µg/ml G-CSF G-CSF 100 µl 8 24 48 72 96 h 20 µl MTT 5 g/l 37 4 h 150 µl DMSO 2 min 490 nm A 3 SPSS10.0 x ± s χ 2 t P < 0.05 HE 3 7 14
2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 173 1 3 6 9 12 15 OP-1 G-CSF 1 OP-1 9 12 15 P < 0.05 G-CSF 9 HE 200 A B C 3 7 14 D E F 3 7 14 Figure 1 Observation of the cells captured by AGS and PLA implanted in mice at different times (HE, original magnification 200). A, B, C respectively showing the cells morphology captured by AGS on days 3, 7, and 14; D, E, F respectively showing the cells morphology captured by PLA on days 3, 7, and 14. Groups Table 1 Quality of AGS treated in different ways after implanted in mice at different times 3 Day 3 6 Day 6 g Quality of AGS (g) 9 Day 9 12 Day 12 15 Day 15 OP-1 OP-1 group 0.1198 0.1171 0.1141 0.1075 0.0872 G-CSF G-CSF group 0.1187 0.1178 0.1104 0.0773 0.0725 Control group 0.1201 0.1168 0.1146 0.1012 0.0921 x ± s Table 2 Number of the cells captured by AGS treated in different ways after implanted in mice at different times ( x ± s ) 10 6 Number of the captured cells ( 10 6 ) 3 6 9 12 15 Groups Day 3 Day 6 Day 9 Day 12 Day 15 OP-1 OP-1 group 0.88 ± 0.04 1.86 ± 0.05 2.91 ± 0.19 a 4.49 ± 0.19 a 5.96 ± 0.22 a G-CSF G-CSF group 0.84 ± 0.02 1.23 ± 0.21 2.36 ± 0.07 a 3.16 ± 0.36 3.81 ± 0.14 Control group 0.71 ± 0.06 1.15 ± 0.13 1.45 ± 0.16 3.11 ± 0.14 3.67 ± 0.85 a P < 0.05 Note: a Compared with control group, P < 0.05.
174 2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 12 CD34 Sca-1 Figure 2 Flow cytometry showing the expression of CD34 and Sca-1 on the cells captured by AGS treated in different ways after implanted in mice on day 12 P < 0.05 12 15 2 OP-1 CD34 Sca-1 13.07% ± 0.19% 3.98% ± 0.15% 17.02% ± 0.12% 11.62% ± 0.44% 3.03% ± 0.11% 2.91% ± 0.14% P < 0.05 G-CSF CD34 Sca-1 12.79% ± 0.39% 4.52% ± 0.35% 10.21% ± 0.15% CD34 Sca-1 P < 0.05 2 MTT OP-1 G-CSF P < 0.05 3 OP-1 G-CSF A490 A490 value 0.8 0.6 0.4 0.2 0 24 48 72 96 h Proliferation time (h) Figure 3 Effects of different culture medium on the proliferative activity of the captured cells [12] [13] G-CSF G-CSF 5 ~ 7 [14] CD34 G-CSF 9 OP-1 OP-1 OP-1 CD34 Sca-1 OP-1
2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 175 OP-1 [1] Brazelton TR, Rossi FM, Keshet GI, et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science, 2000, 290(5497):1775-1779. [2] Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med, 2001, 7(4):393-395. [3] Cao B, Zheng B, Jankowski RJ, et al. Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential. Nat Cell Biol, 2003, 5(7):640-646. [4] Arriero M, Brodsky SV, Gealekman O, et al. Adult skeletal muscle stem cells differentiate into endothelial lineage and ameliorate renal dysfunction after acute ischemia. Am J Physiol Renal Physiol, 2004, 287(4):F621-F627. [5] Qu-Petersen Z, Deasy B, Jankowski R, et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol, 2002, 157(5):851-854. [6] McKinney-Freeman SL, Jackson KA, Camargo FD, et al. Musclederived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci U S A, 2002, 99(3):1341-1346. [7] Tsuboi K, Kawada H, Toh E, et al. Potential and origin of the hematopoietic population in human skeletal muscle. Leuk Res, 2005, 29(3):317-324. [8] Li J, Wang S, Han J, et al. Cells captured from spatium intermusculare by porous material exhibit the characteristics of stem cells. Histochem Cell Biol, 2008, 130(4):741-748. [9] Alessandri G, Pagano S, Bez A, et al. Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages. Lancet, 2004, 364(9448):1872-1883. [10] Wang GD, Wang SL, Han JX. Advances in the study of bone morphogenetic protein-7. J Med Mol Biol, 2004, 1(6):382-385. (in Chinese),,. -7., 2004, 1(6):382-385. [11] Hartung G, Uharek L, Zeis M, et al. Superior antileukemic activity of murine peripheral blood progenitor cell (PBPC) grafts mobilized by G-CSF and stem cell factor (SCF) as compared to G-CSF alone. Bone Marrow Transplant, 1998, 21 Suppl 3:S16-S20. [12] Lu L, Peter SJ, Lyman MD, et al. In vitro degradation of porous poly(l-1actic acid) foams. Biomaterials, 2000, 21(15):1595-1605. [13] Jacobsen S, Fritz HG, Degée P, et al. New developments on the ring opening polymerization of polylactide. Ind Crops Products, 2000, 11(3):265-275. [14] Wang CB, Chen XH, Ou YX, et al. Dynamic changes of CD34~+ cell and T lymphocyte subset from different populations after G-CSF treatment. Acta Acad Medi Milit Tertiae, 2005, 27(12):1269-1271. (in Chinese),,,. G-CSF CD34~+ T., 2005, 27(12): 1269-1271. Study on impact factors of tissue engineering materials capturing adult stem cell DING Ning, WANG Shi-li, HAN Jin-xiang, ZHANG Cui, GE Lu-na Abstract Objective To explore the relevant factors of adult stem cells captured by implanting tissue engineering materials in vivo. Methods BALB/c mice were divided into 2 groups randomly, and respectively implanted absorbable gelatin sponge (AGS) and polymer of lactic acid (PLA) sterilized through ethylene oxide separately in vivo. The AGS and PLA were taken out and observed the captured cells in HE staining on days 3, 7, and 14. BALB/c mice implanted with the same quality of AGS were divided into osteogenic protein-1 (OP-1) group, granulocyte colony-stimulating factor (G-CSF) group, and the control group randomly. The AGS were taken out and weighed, then counted the number of the captured cells on days 3, 6, 9, 12, and 15. The positive rates of CD34 and stem cell antigen-1 (Sca-1) expression on the cells collected on day 12 in each group were detected by flow cytometry. The cells collected on day 12 in the control group were divided into OP-1 group, G-CSF group, and blank control group, and the proliferative activity of the captured cells was detected by MTT. Results HE staining showed that the number of the cells captured by AGS and PLA increased with time prolonged. In each group, the quality of AGS decreased with time prolonged, while the number of the captured cells increased with time prolonged. Compared with the control group, the number of the captured cells in the OP-1 group on days 9, 12, 15 and in the G-CSF group on days 9 were statistically significant (all P < 0.05). Compared with the control group (11.62% ± 0.44%, 3.03% ± 0.11%, and 2.91% ± 0.14% respectively), the single-positive and dual-positive rates of CD34 and Sca-1 expression (13.07% ± 0.19%, 3.98% ± 0.15 %, and 17.02% ± 0.12%, respectively) in the OP-1 group were significantly higher (all P < 0.05), and the single-positive rates of CD34 and Sca-1 expression (12.79% ± 0.39% and 4.52% ± 0.35%) in the G-CSF group were not statistically significant, but the dual-positive rates (10.21% ± 0.15%) were statistically significant (P < 0.05). The absorbance (A) values in the OP-1 group and G-CSF group were
176 2009 6 4 3 Chin Med Biotechnol, June 2009, Vol. 4, No. 3 higher than the blank control group at different times (all P < 0.05). Conclusion Adult stem cells would increased by implanting AGS with OP-1 in vivo, and it could offer evidence to establish a new simple method of capturing auto-stem cell. Key words Adult stem cells; Gelatin sponge, absorbable; Bone morphogenetic proteins; Granulocyte colony-stimulating factor Author Affiliation: Shandong Academy of Medical Sciences, Shandong Medical Biotechnological Center, Key Laboratory for Biotech-Drug of Health Ministry, Key Laboratory for Medical Molecular Biology of Shandong Province, Ji nan 250062, China Corresponding Author: WANG Shi-li, Email: shili2005@sina.com www.cmbp.net.cn Chin Med Biotechnol, 2009, 4(3):171-176 APAB APCB2009 2009 11 6 8 2009 8 31 2009 12 4 6 8 1 PCOS 2 500 ~ 800 4 Word 41 100032 qinjietn@sohu.com 2009 10 20