ABSTRACT
AIM:To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process .METHODS:MSCs were isolated and cultured in vitro.The cells were divided into LiCl group and control group .MSCs were treated withβ-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons .The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associ-ated protein 1 light chain 3 ( LC3) were measured by immunofluorescence method and Western blot .An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells.The protein expression of NSE and MAP-2 were determined by Western blot .RESULTS: After induction, the ex-pression of NSE and MAP-2 were increased .The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05).After induction, the number of LC3-positive dots and the expression of LC3-Ⅱin LiCl group were greater than those in control group (P<0.05).The expres-sion of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated -cells, and on the contra-ry, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA.CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy .
ABSTRACT
The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.
Subject(s)
Humans , Antigens, CD34 , Bone Marrow Cells , Cell Biology , Allergy and Immunology , Cell Culture Techniques , Methods , Cell Differentiation , Cell Division , Cell Separation , Cells, Cultured , Erythroid Precursor Cells , Cell Biology , Fetal Blood , Cell Biology , Allergy and Immunology , Megakaryocyte Progenitor Cells , Cell Biology , Allergy and ImmunologyABSTRACT
This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.
Subject(s)
Humans , Antigens, CD34 , Allergy and Immunology , Cell Differentiation , Cell Division , Cell Separation , Cells, Cultured , Fetal Blood , Cell Biology , Allergy and Immunology , Megakaryocyte Progenitor Cells , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the blood supply and the clinical application of the posterior calf fasciocutaneous flap for repairing the frontal defect of knee joint.</p><p><b>METHODS</b>Based on the article review and the anatomical study of the posterior calf in 8 cadavers (16 sides), 10 cases with frontal defects of knee joints were repaired with island fasciocutaneous flaps which had pedicles of lateral superficial sural artery and the lateral sural nerve.</p><p><b>RESULTS</b>The anatomical study showed there were three systems of superficial sural blood supply- medial, middle and lateral systems. They are originated from popliteal artery or from the bilateral sural artery. The lateral superficial sural artery was present in 100% of the cadavers. The flaps survived completely in 9 cases, except one case with partial necrosis at the one-fourth distal end of the flap. The patients were followed up for 6-12 months with good aesthetic and functional results.</p><p><b>CONCLUSIONS</b>The posterior calf island fasciocutaneous flap has an reliable blood supply and protective sense nerve. The flap is ideal for the reconstruction of soft tissue defect around knee joint.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Male , Young Adult , Leg , General Surgery , Muscle, Skeletal , Transplantation , Skin Transplantation , Methods , Surgical FlapsABSTRACT
<p><b>OBJECTIVE</b>The nose is composed of several delicate subunits, some of which are difficult to reconstruct if they have been injured. The paper presents the microsurgical technique to repair the nasal subunit defects with the free combined preauricular and auricular flap which well match the nasal tissues in texture, contour and color.</p><p><b>METHODS</b>The nasal subunit defects were repaired with the combined preauricular and auricular flap which were vascularized by the superficial temporal vascular system. The flap was harvested from the contralateral preauricular and the region of helix crus. The superficial temporal vessels were anastomosed to facial vessels via the vascular grafts harvested from the lateral circumfluent femoral vessels, which were about 10 to 14 cm in length. The helix crus of donor ear was reconstructed with the post-auricular flap.</p><p><b>RESULTS</b>28 cases were treated, including 3 cases of nasal tip defects, 9 cases of combined nasal alar and sidewall defects, and 16 cases of nasal alar defects. In these cases, the size of the subunit defects varied from 2.5 cm x 1.5 cm to 4 cm x 2.5 cm. 27 cases were successfully repaired with satisfactory results. The contour of reconstructed helix crus in donor site was acceptable. No walking dysfunction of the donor thigh was complained. There is one failed case, and the possible reason is the insufficient blood perfusion to the flaps, which may due to the patient's longtime-smoking status and the hypertension.</p><p><b>CONCLUSIONS</b>The technique of free combined preauricular and auricular flap is ideal for the reconstruction of nasal subunit defects.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Ear Auricle , General Surgery , Microsurgery , Nose , Congenital Abnormalities , Nose Deformities, Acquired , General Surgery , Rhinoplasty , Methods , Skin Transplantation , Surgical FlapsABSTRACT
<p><b>OBJECTIVE</b>To assess and review the indications and methods of the reversed posterior interosseous artery flap for repairing soft tissue defects of the hand.</p><p><b>METHODS</b>In the series, three types of flaps were utilized including the reversed posterior interosseous artery flap, the expanded reversed posterior interosseous artery flap and the reversed posterior interosseous artery conjoint flap. 78 clinical cases were treated with this method, in which 12 cases used the expanded reversed posterior interosseous artery flap and 10 cases used the reversed posterior interosseous artery conjoint flap. The flap,ranged from 8 approximately 6 cm to 14 approximately 8 cm, was used to repair the defects of the dorsal hand as far as to the fingerweb. The anatomy of the posterior interosseous artery was observed in the surgical dissection.</p><p><b>RESULTS</b>The posterior interosseous artery was consistently identified in all the 78 cases. In 4 cases, the artery terminated in the middle third of the dorsal forearm, therefore an alternative method of reconstruction was used. In the 53 cases of reversed posterior interosseous flaps, 49 cases healed uneventfully and 4 cases suffered necrosis in the distal border of the flap, but subsequently healed spontaneously. 12 cases of expanded flaps survived well and the donor sites were directly approximated. In the 9 cases of conjoint flaps based on the posterior interosseous artery, 7 cases healed uneventfully and 2 cases suffered delayed healing because of flap necrosis of the distal part. The satisfactory appearance and hand function were achieved postoperatively in all cases.</p><p><b>CONCLUSIONS</b>The flap is a better choice for repairing the soft tissue defects of the hand. The different type of flaps can be selected to meet the clinical requirements of reconstruction. It is suggested that the artery variation should be assessed preoperatively.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Arteries , Transplantation , Finger Injuries , General Surgery , Head , General Surgery , Retrospective Studies , Surgery, Plastic , Methods , Surgical Flaps , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>Despite the causes for melanin increase, the increased gene expression of TYR is a common pathological process. Based on this viewpoint, antisense-S-Oligo of TYR was designed and synthesized to regulate synthesis of melanin in order to explore the treatment for skin pigmentation.</p><p><b>METHODS</b>The cultured melanocytes were divided into 3 groups. The group 1 was treated with endothelin, group 2 treated with ultraviolet ray and group 3 was used as the control. In each group, the 5' antisense-S-Oligo, the 3' antisense-S-Oligo, the mixed antisense-S-Oligo of TYR or Dotap only was added. The melanin content and TYR gene expressions were examined.</p><p><b>RESULTS</b>The 5' antisense-S-Oligo, the 3' antisense-S-Oligo and the mixed antisense-S-Oligo significantly inhibited the increase of melanin content and TYR gene expression, which were caused by endothelin or ultraviolet ray treatment. Of the three treatments, the 3' antisense-S-Oligo showed the strongest effect.</p><p><b>CONCLUSION</b>Antisense-S-Oligo has significant regulating effects on TYR gene expression and melanin content. The 3' antisense-S-Oligo is more effective than the 5' antisense-S-Oligo.</p>