RA induces the neural-like cells generated from epigenetic modified NIH/3T3 cells.

Recently, differentiated somatic cells had been reprogrammed to pluripotential state in vitro, and various tissue cells had been elicited from those cells. Epigenetic modifications allow differentiated cells to perpetuate the molecular memory needed for the cells to retain their identity. DNA methylation and histone deacetylation are important patterns involved in epigenetic modification, which take critical roles in regulating DNA expression. In this study, we dedifferentiated NIH/3T3 fibroblasts by 5-aza-2-deoxycytidine (5-aza-dC) and Trichstatin A (TSA) combination, and detected gene expression pattern, DNA methylation level, and differentiation potential of reprogrammed cells. As the results, embryonic marker Sox2, klf4, c-Myc and Oct4 were expressed in reprogrammed NIH/3T3 fibroblasts. Total DNA methylation level was significant decreased after the treatment. Moreover, exposure of the reprogrammed cells to all trans-retinoic acid (RA) medium elicited the generation of neuronal class IIIbeta-tubulin-positive, neuron-specific enolase (NSE)-positive, nestin-positive, and neurofilament light chain (NF-L)-positive neural-like cells.

MSCs guide neurite directional extension and promote oligodendrogenesis in NSCs.

Previous studies have shown that mesenchymal stem cells (MSCs) enhance repair following injury or degenerative diseases in the central nervous system, but the underlying mechanisms remain unclear. The present study investigated the functional relationship between MSCs and neural stem cells (NSCs) using co-culture systems. Results demonstrated that MSCs promoted outgrowth and guided directional extension of NSC-derived neurites. The majority of neurites were oriented parallel along the MSC axis. Stripe assay results indicated that cell adhesion molecule and extracellular matrix, such as N-cadherin, fibronectin, and laminin, contributed to this effect. Furthermore, Western blot analysis revealed that phosphorylation of cAMP response element-binding protein (CREB) increased during this process. In addition, MSCs promoted differentiation of NSCs into oligodendrocytes via secreted soluble factors. The oligodendrocytes were distributed along the MSC surface in a regular pattern. This study demonstrated that MSC transplantation could be a potential strategy for treating central nervous system injuries.

[Effects of 4-hydroxytamoxifen on prostate smooth muscle cells: an in vitro experiment].

OBJECTIVE: To investigate the effects of 4- hydroxytamoxifen (OHT) on the proliferation and apoptosis of prostate smooth muscle cells and the expression of estrogen receptor (ER) and androgen receptor (AR). METHODS: Prostate smooth muscle cells were isolated from the resected specimens of prostate glands of 10 patients with benign prostatic hypertrophy (BPH), cultured, and exposed to estradiol (E(2)), diethylstilbestrol (DES), and OHT of different concentrations (1 x 10(-8) - 1 x 10(-5) mol/L) or mixture of E(2) (1 x 10(-8) - 1 x 10(-6) mol/L) with OHT (1 x 10(-7) mol/L). Flow cytometry was used to test the proliferation and apoptosis of the cells, and immunocytochemistry was used to test the expression of estrogen and androgen receptors. RESULTS: E(2) and DES promoted the proliferation of the prostate smooth muscle cells in a certain concentration range, but not dose-dependently, and OHT at the concentration of 1 x 10(-8) mol/L slightly increased the G(2)-M peak rate of the prostate smooth muscle cells, but suppressed the G(2)-M peak rate dose-dependently when its concentration was >or= 1 x 10(-7) mol/L (P < 0.05) and this suppression effect was dose-dependently (r = -0.312, P = 0.011). E(2) at the concentration >or= 1 x 10(-5) mol/L and DES at the concentration >or= 1 x 10(-6) mol/L slightly promoted the apoptosis of the prostate smooth muscle cells, but not dose-dependently, and OHT at the concentrations from 1 x 10(-8) mol/L to 1 x 10(-5) mol/L promoted the apoptosis of the prostate smooth muscle cells dose-dependently (r = 0.363, P = 0.021) and this effect could not be reversed by administration of E(2) at the concentration 1 x 10(-8) - 1 x 10(-6) mol/L (P > 0.05). E(2), DES, and OHT of different concentrations all increased the ERalpha and AR positive staining rates of the prostate smooth muscle cells (all P < 0.05). CONCLUSIONS: OHT suppresses the proliferation and promotes the apoptosis of prostate smooth muscle cells, and these functions do not depend on the estrogen receptor pathway. Low blood OHT concentration after oral administration of TAM and up-regulation of estrogen receptors by OHT may be the caused of the inefficiency of TAM for treatment of BPH.

[Effects of 40H-tamoxifen on the proliferation and apoptosis of prostate stromal cells].

OBJECTIVE: To investigate the effects of 4OH-Tamoxifen (OHT) on proliferation and apoptosis of primary cultured prostate stromal cells. METHODS: Primarily cultured prostate stromal cells in vitro were treated with various concentrations (10(-8) mol/L - 10(-5) mol/L) of estradiol (E2), diethylstilbestrol (DES), OHT and the mixture of E2 (10(-8) mol/L - 10(-6) mol/L) with OHT (10(-7) mol/L) and then MTT and TUNEL were used to detect their proliferation and apoptosis respectively. RESULTS: There was a significant difference (P < 0.05) between OHT and estrogens in the effects on the apoptosis and proliferation of the primarily cultured prostate stromal cells. OHT suppressed proliferation of the prostate stromal cells at the concentrations from 10(-7) mol/L to 10(-5) mol/L (P < 0.05), and this effect was concentration related (r = -0.383, P = 0.005); OHT (10(-7) mol/L) suppressed the proliferation stimulation effect of E2 at the concentrations from 10(-8) mol/L to 10(-6) mol/L (P < 0.05). OHT induced apoptosis at the concentrations from 10(-8) mol/L to 10(-5) mol/L (P < 0.05), and this effect was concentration related (r = 0.349, P = 0.012). The apoptosis induced by OHT could not be reversed by E2 at the concentrations from 10(-8) mol/L to 10(-5) mol/L (P > 0.05). CONCLUSION: OHT can obviously suppressed the proliferation and promote the apoptosis of primarily cultured prostate stromal cells, which might not be totally attributed to the competitive inhibition of the estrogen receptor.

[Tissue culture of bovine lens as an in vitro model for posterior capsule opacification and the effects of pranoprofen on the cell confluence].

OBJECTIVE: To establish an in vitro model of posterior capsule opacification (PCO) by culturing the posterior capsule of bovine lens, to observe the proliferation and differentiation of lens epithelial cells and to study the influence of serum and pranoprofen eyedrops on cell confluence of this model. METHODS: The bovine lens posterior capsule was spread on the surface of a 25 ml culture flask with cell layer upward. DMEM with 0%, 10% and 20% fetal calf serum was used as culture medium. The cell coverage and confluence time on the posterior capsule were observed by inverted microscope and the cell morphology was observed by Giemsa staining and scanning electron-microscope. Pranoprofen was added to the culture medium at a concentration similar to the aqueous humor concentration (0.23 mg/L), which was presented at 4 hours after the instillation of pranoprofen eyedrops. The difference of confluence time between the treated group and the control group was compared. RESULTS: The lens epithelial cells migrated and proliferated rapidly on the posterior capsule from the equatorial region to the center. The cell coverage was increased and the confluence time was shortened with the increase of serum concentration (P < 0.05). The PCO and wrinkles were presented. Pranoprofen at 0.23 mg/L could inhibit the confluence of lens epithelial cells (P < 0.01). CONCLUSIONS: The in vitro model for PCO was an useful method to study the mechanism of PCO formation. Pranoprofen can inhibit the proliferation of lens epithelial cells and is a safe and efficient drug for preventing the occurrence of PCO and can be used as a routine medication after the cataract operation.

4-Hydroxynonenal Regulates TNF-α Gene Transcription Indirectly via ETS1 and microRNA-29b in Human Adipocytes Induced From Adipose Tissue-Derived Stromal Cells.

Obesity is characterized by an accumulation of excessive body fat and can be diagnosed by a variety of measures, such as BMI. However, in some obese individuals, oxidative stress is also thought to be an important pathogenic mechanism of obesity-associated metabolic syndrome. Oxidative stress increases the lipid peroxidation product, 4-hydroxynonenal (4-HNE), which is one of the most abundant and active lipid peroxides. Within the adipose tissue, adipocytes are derived from adipose tissue-derived stromal cells (ADSCs), which play a key role in the generation and metabolism of adipose tissue. Additionally, obesity is associated with low-grade inflammation. Specific microRNAs (miRNAs) that regulate obesity-associated inflammation are largely dysregulated in metabolic syndrome (MS). In this study, we aim to confirm whether 4-HNE and miRNAs play a role in the regulation of TNF-α gene transcription. We enrolled six obese individuals who were referred to Harbin Medical University (Heilongjiang, China) and six nonobese control participants. Plasma 4-HNE levels of the 12 subjects were determined by ELISA. Using qRT-PCR, we measured ETS1, miR-29b, SP1, and TNF-α levels in subcutaneous white adipose tissue (WAT). Furthermore, we examined the relationship between ETS1 and TNF-α using a luciferase reporter assay and a ChIP assay. Our results suggest that ETS1 promotes TNF-α gene transcription in adipocytes. In addition, we demonstrated that 4-HNE promotes TNF-α gene transcription through the inhibition of the miR-29b â†’ SP1 â†’ TNF-α pathway and promotion of the ETS1 â†’ TNF-α pathway. Anat Rec, 299:1145-1152, 2016. © 2016 Wiley Periodicals, Inc.

MicroRNA-29b promotes the adipogenic differentiation of human adipose tissue-derived stromal cells.

OBJECTIVE: Obesity is primarily characterized by the accumulation of large amounts of fat in adipose tissue. Within the adipose tissue, adipocytes are derived from adipose tissue-derived stromal cells (ADSCs) via a specialized cell lineage differentiation process, and ADSCs play a key role in the generation and metabolism of adipose tissue. This study investigated whether microRNAs (miRNAs) play a role in adipocyte differentiation. METHODS: Using luciferase reporter and ChIP assays, the relationship between miR-29b, SP1, and TNF-α was examined. RESULTS: During the normal adipogenic differentiation of ADSCs, up-regulation of miR-29b promoted adipogenesis by enhancing SP1-mediated inhibition of TNF-α. CONCLUSIONS: This study investigated the regulatory role of miR-29b during the adipogenic differentiation of ADSCs and found that miR-29b is an effective positive regulator of adipogenesis.

Posterior scleral reinforcement for the treatment of pathological myopia.

AIM: To investigate the effects of posterior scleral reinforcement (PSR) in the treatment of pathological myopia. METHODS: The study included 52 eyes in 43 patients with pathological myopia who underwent PSR (PSR group), and 52 eyes in 36 age- and myopia-matched patients who did not undergo such treatment as control group. Axial length, refraction error, best corrected visual acuity (BCVA), and macular scans by optical coherence tomography (OCT) were recorded at baseline, 6mo, 1, 3 and 5y after the surgery, and the complications were noted. RESULTS: There were no statistical differences in axial length, refractive error, or BCVA between the PSR group and the control group at baseline. At the end of the follow-up, the mean axial length was 29.79±1.26 mm in the PSR group, which was significantly shorter than that in the control group (30.78±1.30 mm) (P<0.01), and the mean refractive error was -16.86±2.53 D in the PSR group, which was significantly lower than that in the control group (-19.18±2.12 D) (P<0.01). A statistically significant difference in BCVA was found between the PSR group (0.51±0.25 logMAR) and the control group (0.62±0.26 logMAR) at the postoperative 5-year follow-up (P<0.01). There were no serious complications during the 5-year follow-up period. CONCLUSION: PSR can prevent axial elongation and myopia progression in eyes with pathological myopia.

Posterior scleral reinforcement combined with vitrectomy for myopic foveoschisis.

AIM: To investigate the effects of posterior scleral reinforcement (PSR) combined with vitrectomy for myopic foveoschisis. METHODS: Thirty-nine highly myopic eyes of 39 patients with myopic foveoschisis underwent PSR combined with vitrectomy. Best corrected visual acuity (BCVA), refraction error, and the foveal thickness by optical coherence tomography (OCT) were recorded before and after the surgery, and complications were noted. RESULTS: The follow-up period was 12mo, and the main focus was on the results of the 12-month follow-up visit. The mean preoperative BCVA was 0.96±0.43 logMAR. At the final follow-up visit, the mean BCVA was 0.46±0.28 logMAR, which significantly improved compared with the preoperative one (P=0.003). The BCVA improved in 33 eyes (84.62%), and unchanged in 6 eyes (15.38%). At the end of follow-up, the mean refractive error was -15.13±2.55 D, and the improvement was significantly compared with the preoperative one (-17.53±4.51 D) (P=0.002). Twelve months after surgery, OCT showed complete resolution of the myopic foveoschisis and a reat-tachment of the fovea in 37 eyes (94.87%) and partial resolution in the remained two eyes (5.13%). The foveal thickness was obviously reduced at 12-month follow-up visit (196.45±36.35 µm) compared with the preoperative one (389.32±75.56 µm) (P=0.002). There were no serious complications during the 12mo follow-up period. CONCLUSION: PSR combined with vitrectomy is a safe and effective procedure for myopic foveoschisis with both visual and anatomic improvement.

Ranibizumab Plus Combined Surgery for Treatment of Neovascular Glaucoma with Vitreous Hemorrhage.

BACKGROUND: Neovascular glaucoma (NVG) is a refractory glaucoma. The management of NVG is very difficult, and it is more difficult when combined with vitreous hemorrhage. The aim of this study was to investigate the effects of ranibizumab plus combined surgery for NVG with vitreous hemorrhage. METHODS: A total of 26 eyes of 26 NVG patients with vitreous hemorrhage were recruited in this study. The patients aged from 36 to 63 years with a mean age of 51.97 ± 7.60 years. The mean intraocular pressure (IOP) was 46.38 ± 5.75 mmHg (1 mmHg = 0.133 kPa) while being treated with the maximum medical therapy. The mean best-corrected visual acuities converted to logarithm of the minimum angle of resolution (logMAR BCVA) was 2.62 ± 0.43. All the patients underwent intravitreal injection of 0.5 mg (0.05 ml) ranibizumab combined with pars plana vitrectomy (PPV), pars plana lensectomy (PPL) with a preserved anterior capsule, panretinal photocoagulation (PRP), and trabeculectomy (intravitreal ranibizumab [IVR] + PPV + PPL + PRP + trabeculectomy). The IOP and logMAR BCVA were the main outcome measures in this study. RESULTS: The follow-up period was 12 months. The mean postoperative IOPs were 26.38 ± 3.75 mmHg, 21.36 ± 3.32 mmHg, 18.57 ± 3.21 mmHg, and 16.68 ± 2.96 mmHg, respectively at 7 days, 1 month, 3 months, and 12 months after PPV + PPL + PRP + trabeculectomy. At the last follow-up, the mean IOP was significantly lower than the preoperative one (t = 6.612, P = 0.001). At 7 days, 1 month, 3 months, and 12 months after PPV + PPL + PRP + trabeculectomy, the mean logMAR BCVA were 1.30 ± 0.36, 1.29 ± 0.37, 1.29 ± 0.39, and 1.26 ± 0.29, respectively. At the last follow-up, the mean logMAR BCVA was significantly improved, and the difference was statistically significant compared with preoperative one (t = 6.133, P = 0.002). The logMAR BCVA improved in 22 eyes (84.62%), and remained stable in 4 eyes (15.38%). The neovascularization in the iris and the angle regressed significantly in all patients 7 days after ranibizumab injection. No serious complications occurred during 12 months of the study. CONCLUSIONS: IVR + PPV + PPL + PRP + trabeculectomy can control IOP well and improve BCVA without severe complication for NVG patients with vitreous hemorrhage.

Generation of dorsal spinal cord GABAergic neurons from mouse embryonic stem cells.

Developmental signaling molecules involved in dorsal patterning of the spinal cord have been identified in vivo; however, studies have not produced specific functional dorsal spinal cord neurons in vitro. We present here differentiation of R1 embryonic stem (ES) cells into GABAergic dorsal spinal cord neurons by sequential treatment with developmental signaling molecules. We found that retinoic acid, Bmp4 altered the specification of neural progenitors and instructed neural fate when applied at distinct stages of development. High concentration of retinoic acid initiated caudal patterning during early differentiation; Bmp4 induced dorsal development. The combination of retinoic acid and different concentration Bmp4 controlled the differing regional progenitors of spinal cord. Low-concentration Bmp4 and high concentration of retinoic acid-treated embryoid bodies resulted in the differentiation of GABAergic neurons. In summary, we demonstrate this simple treatment paradigm produced simple dorsal spinal cord neurons, which could be utilized for developmental and preclinical studies.

pCREB is involved in neural induction of mouse embryonic stem cells by RA.

Mouse embryonic stem (ES) cells can be induced by various chemicals to differentiate into a variety of cell types in vitro. In our study, retinoic acid (RA), one of the most important inducers, used at a concentration of 5 microM, was found to induce the differentiation of ES cells into neural progenitor cells (NPCs). During embryoid body (EB) differentiation, the level of active cyclic AMP response element-binding protein (CREB) was relatively high when 5 microM RA treatment was performed. Inhibition of CREB activity committed EBs to becoming other germ layers, whereas increased expression of CREB enhanced NPC differentiation. Moreover, RA increased the expression of active CREB by enhancing the activity of JNK. Our research suggests that CREB plays a role in RA-induced NPC differentiation by increasing the expression of active JNK.

[Culture of porcine mesenchymal stem cells in vitro and developmental capacity of reconstructed embryos by nuclear transfer].

The developmental potential of reconstructed embryos varied according to the source of donor cells, it was thought that the donor cells capabilities to be reprogrammed were different. We established the method of culturing porcine bone marrow mesenchymal stem cells (pMSCs), identified and observed the growth characteristics of pMSCs, and determined pMSCs reprogramming potential as donor cells for nuclear transfer (SCNT). We found that the method of gradient centrifugation to isolate pMSCs from porcine bone marrow was better than the method of anchoring culture; the number of pMSCs achieved peak at day 6, the adhesive rate of cultured cells was 78.50% at 10h and the division index of cultured cells was 24.00 per thousand at day 4. The developmental competence were compared among three kinds of embryos, reconstructed embryos with PF and pMSCs, Parthenogenetic. The blastocysts rate and total cell number of blastocysts were 15.07%, 14.63% vs 30.91% and 24.1 +/- 6.5, 30.67 +/- 17.7 vs 25.8 +/- 11.4. These results indicated that pMSCs could be high proliferation and stable growth characters in vitro, and were suitable donor cells type for nuclear transfer.

Carrier screening for alpha- and beta-thalassemia in pregnancy: the results of an 11-year prospective program in Guangzhou Maternal and Neonatal hospital.

OBJECTIVES: To evaluate the first prospective screening program in China for control of alpha and beta-thalassemia in the population of pregnant couples. METHODS: During the period between January 1993 and December 2003, a hospital-based preventive program was conducted at the biggest birth center in Guangzhou, with 1/17 of all deliveries in this city referred annually by use of conventional heterozygote screening strategy in combination with the system of regular healthcare examination in pregnancy. RESULTS: The screened records included 49 221 pregnant women, and 4503 husbands of the pregnant women showed positive on the screening test. Of the at-risk couples, there were 198 for alpha-thal (4.4%) and 83 for beta-thal (1.8%), respectively. Genetic counseling was offered to all at-risk couples and a successful prenatal diagnosis was performed for 269 out of 281 (95.7%) for alpha- or beta-thal major, with the remaining 12 couples refusing to accept prenatal diagnosis. Out of 187 pregnancies at risk for homozygous alpha0-thal and 82 at risk for beta-thal major, 51 hydrops fetalis with Hb Bart's and 18 beta-thal major were identified. All pregnancies with affected fetuses were voluntarily terminated, leading to a marked reduction of severe alpha- and beta-thal births at this hospital since the program has been launched. CONCLUSIONS: Our hospital-based program proved to be highly effective in reducing severe thals in pregnant populations.