[Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds].

Objective: To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods: Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results: Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions: Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.

[Feasibility of radiofrequency ablation for cancer patients with atrial fibrillation].

Objective: To analyze the impact of cancer on the recurrence rate of atrial fibrillation (AF) after AF radiofrequency ablation and further evaluate the feasibility of radiofrequency ablation therapy in cancer patients with AF. Methods: This study was a single-center, retrospective study. Cancer patients with AF undergoing radiofrequency ablation for the first time in the First Affiliated Hospital of Dalian Medical University from May 30, 2008 to September 30, 2018 were included (cancer group). AF patients without cancer undergoing radiofrequency ablation for the first time during the same period served as non-cancer group. Clinical data including age, gender, past history, cancer and AF-related parameters, etc. were analyzed. Patients were followed up after radiofrequency ablation. The primary endpoints were AF recurrence or all-cause death. Kaplan-Meier survival analysis was used to analyze the effect of cancers on the recurrence after AF ablation. The multivariate cox regression analysis was further applied to correct for other confounding factors to analyze whether the impact of cancers on the recurrence of atrial fibrillation was statistically significant. Results: A total of 90 patients were enrolled, there were 30 patients in the cancer group (mean age (64.8±6.6) years, 16 (53.3%) males) and 60 patients in the non-cancer group (mean age (63.6±6.2) years, 32 (53.3%) males). Clinical data, such as age, gender, and cancer treatment, were similar between the two groups. During an average follow-up period of (328.7±110.2) days, there were 6 AF recurrences (recurrence rate 20.0%) in the cancer group, and 17 AF recurrences (recurrence rate 28.3%) in the control group. AF recurrence rate was similar between the two groups (P>0.05). During the follow-up period, there was no all-cause death in the two groups. Kaplan-Meier survival analysis showed that cancer was not related to AF recurrence after radiofrequency ablation (P = 0.383). After adjusting for other confounding factors, the multivariate Cox regression analysis showed that cancer was not an independent predictor of AF recurrence after radiofrequency ablation (HR=0.508, 95%CI: 0.192-1.342, P = 0.172). Conclusions: The combination of cancer has no impact on the recurrence of AF after radiofrequency ablation. For cancer patients with AF, radiofrequency ablation therapy can be considered as a feasible heart rhythm control treatment strategy.

Long non-coding RNA GClnc1 promotes progression of colorectal cancer by inhibiting p53 signaling pathway.

OBJECTIVE: The aim of this study was to investigate whether long non-coding RNA (lncRNA) GClnc1 was involved in the development of colorectal cancer, and to explore its possible mechanisms. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was conducted to detect GClnc1 expression in 48 colorectal cancer tissues and normal colon tissues. The Kaplan-Meier method was used to analyze the relationship between GClnc1 expression and survival rate of patients with colorectal cancer. In addition, GClnc1 expression in colorectal cancer cell lines and normal colonic epithelial cell lines were analyzed. After knockdown and over-expression of GClnc1 in colorectal cancer cells, Cell Counting Kit-8 (CCK-8) and colony formation assay were performed to detect the viability and proliferation of cells, respectively. RNA pull-down and RNA-binding protein immunoprecipitation (RIP) were applied to examine the specific interaction between GClnc1 and p53. After over-expression of GClnc1 in colorectal cancer cells, qPCR and Western blot were performed to evaluate the expression levels of p53, p21 and BAX. Meanwhile, the Luciferase reporter gene assay was established to reveal the activity of p53 after over-expression of GClnc1. ChIP assay was applied to figure out whether GClnc1 could affect the binding ability of p53 to the promoter region of p21. After p53 or GClnc1 knock-down in colorectal cancer cells, the protein level of p53 was analyzed using Western blot. Finally, qRT-PCR, CCK-8 and colony formation assay were used to detect the levels of p21 and BAX, the viability, as well as the proliferation ability of cells, respectively. RESULTS: The expression of GClnc1 in colorectal cancer tissues was significantly higher than that of para-cancerous tissues. Meanwhile, GClnc1 expression in T3 and T4 tumors was markedly higher than that of T1 and T2. The survival analysis revealed that patients with a higher level of GClnc1 showed remarkably lower overall survival than those with lower expression of GClnc1. QRT-PCR results indicated that GClnc1 expression in colorectal cancer cells (including SW620 and HCT116) was conspicuously higher than that of normal colonic epithelial cells (NCM640). After knocking down GClnc1 in SW620 cells, the viability and proliferation abilities were conspicuously decreased. Meanwhile, the expression level of GClnc1, as well as the viability and colony formation ability of cells, were significantly increased after over-expression of GClnc1 in HCT116 cells. Subsequently, the qRT-PCR assay demonstrated that GClnc1 was mainly localized in the nucleus. RNA pull-down and RIP experiments revealed that there was a specific interaction between GClnc1 and p53. Moreover, qRT-PCR and Western blot analysis indicated that the expression level of p53 was not affected after over-expression of GClnc. However, the expressions of p21 and BAX were remarkably decreased. The Luciferase reporter gene assay revealed that GClnc1 over-expression markedly weakened the Luciferase activity of p53. Meanwhile, ChIP experiments demonstrated that GClnc1 up-regulation affected the binding condition of p53 to p21. Western blot analysis showed that knockdown of p53 reversed the increased mRNA level of p21 as well as BAX. Furthermore, p53 down-regulation significantly weakened cell viability and colony formation ability caused by knockdown of GClnc1. CONCLUSIONS: LncRNA GClnc1 was highly expressed in colorectal cancer tissues. Meanwhile, it could increase the proliferation of colorectal cancer cells by reducing the expression of p21 as well as BAX via p53 signaling pathway, thereby promoting the progression of colorectal cancer.

A pilot split-scalp study of combined fractional radiofrequency microneedling and 5% topical minoxidil in treating male pattern hair loss.

BACKGROUND: Various trials have been conducted on the management of male pattern hair loss (MPHL), but the outcomes often seem to be limited. Adjuvant therapies are urgently needed. AIM: To evaluate the efficacy and safety of combined fractional radiofrequency microneedling (FRM) and 5% topical minoxidil in the treatment of male pattern hair loss. METHODS: In total, 19 Chinese men were enrolled in this randomized, controlled, split-scalp trial. Participants received monotherapy with 5% topical minoxidil twice daily to one half of the scalp, while on the other half of the scalp the treatment with twice-daily 5% topical minoxidil was combined with five sessions of FRM at 4-week intervals. Mean hair count and hair thickness, global assessment by the investigators, subject self-assessment and adverse effects were assessed. RESULTS: After 5 months of treatment, mean hair count increased from 44.12 ± 21.58 to 73.14 ± 25.45 on the combined-therapy side and from 46.22 ± 18.77 to 63.21 ± 19.22 on the monotherapy side, while mean hair thickness increased from 53 ± 13 µm to 71 ± 15 µm and from 52 ± 16 µm to 66 ± 14 µm, respectively. Compared with the monotherapy side, the combined-therapy side had a higher degree of improvement in both hair count (P = 0.01) and hair thickness (P = 0.02). CONCLUSIONS: Combined treatment with fractional radiofrequency microneedle and 5% topical minoxidil could be an effective and safe treatment option for male pattern hair loss.

Acetal-linked branched poly(dimethyl-aminoethyl methacrylate) as an acid cleavable gene vector with reduced cytotoxicity.

An acid labile branched PDMAEMA/acetal copolymer with amino group was synthesized by the DE-ATRP and followed by Michael addition. The degradation of the polymer was strongly pH-dependent. High nucleic acid transfection efficiency with low cytotoxicity was observed compared to its non-degradable copolymer counterpart.

CT findings of anomalous systemic artery to the left lower lobe: comparison with pulmonary sequestration in the left lower lobe.

AIM: To analyse and compare CT findings of anomalous systemic artery to the left lower lobe (ASALLL) and pulmonary sequestration (PS) in the left lower lobe (LLL). MATERIALS AND METHODS: The present study cohort comprised 16 patients with ASALLL and 25 patients with PS in the LLL confirmed by surgical and pathological findings. Medical records and CT images were reviewed retrospectively. RESULTS: Cough and sputum were more common in PS (84% and 60%, respectively) than in ASALLL (25% and 12.5%, respectively; p < 0.05). Haemoptysis was more common in ASALLL (100%) than in PS (24%; p < 0.05). The frequency of ground-glass opacity (GGO), normal bronchial distribution, dilated left inferior pulmonary veins, and absence of the interlobar artery distal to the origin of the superior segmental artery in the LLL differed significantly between ASALLL and PS. A mass was less common in ASALLL (0%) than in PS (88%; p < 0.01). The mean diameters of the anomalous artery (12.10 ± 1.49 mm) and left inferior pulmonary veins (13.20 ± 2.19 mm) in ASALLL were significantly larger than those (6.73 ± 2.59 mm, 10.04 ± 1.55 mm) in PS. The threshold diameters of the anomalous artery and left inferior pulmonary vein for ASALLL were 9.75 and 11.75 mm, respectively. The presence of an anomalous artery arising from the thoracic aorta was not different between ASALLL (100%) and PS (72%). CONCLUSION: The following imaging features favour ASALLL over PS in the LLL: a larger anomalous systemic artery arising from the thoracic aorta, dilated left inferior pulmonary veins, absence of the interlobar artery distal to the origin of the superior segmental artery, normal bronchial distribution, and GGO in the LLL.

The effect of remifentanil on the incidence of agitation on emergence from sevoflurane anaesthesia in children undergoing adenotonsillectomy.

The aim of the present study was to assess the effect of remifentanil on the incidence of emergence agitation in preschool-aged children undergoing adenotonsillectomy with sevoflurane anaesthesia. Sixty children, aged three to seven years, American Society of Anesthesiologists physical status I or II, were randomised into either group S (sevoflurane alone, n=30) or group R (sevoflurane plus remifentanil, n=30). Anaesthesia was induced with an intravenous bolus injection of fentanyl 3 microg/kg and propofol 2.5 mg/kg. Endotracheal intubation was facilitated by vecuronium 0.1 mg/kg. All patients were ventilated with 50% nitrous oxide and 1.5 to 2.5% sevoflurane in oxygen. End-tidal CO2 was maintained at 35 +/- 4 mmHg. Group S received no other medication while group R received remifentanil 1 microg/kg/minute intraoperatively. Mean blood pressure, heart rate, pulse oximetry, eye-opening time and extubation time were recorded in the operating room. In recovery, emergence agitation was assessed using the Pediatric Anesthesia Emergence Delirium scale with a score > or =10 taken as indicating agitation. Emergence agitation occurred in 20 of the 30 patients in group S and seven of the 30 patients in group R (P < 0.01). In preschool-aged children undergoing adenotonsillectomy with sevoflurane general anaesthesia, after propofol and fentanyl induction, intraoperative remifentanil decreased the incidence of emergence agitation.

Synaptic connections between trigemino-parabrachial projection neurons and gamma-aminobutyric acid- and glycine-immunoreactive terminals in the rat.

The synaptic connections between gamma-aminobutyric acid (GABA)- and glycine-immunoreactive terminals and neurons projecting to the lateral parabrachial region were examined by a combination of retrograde tracing and immunohistochemical staining in the rat medullary dorsal horn. After injection of horseradish peroxidase (HRP) into the right lateral parabrachial region, HRP retrogradely labeled neurons were observed bilaterally in laminae I, II and III of the medullary dorsal horn with an ipsilateral predominance. GABA- and glycine-like immunoreactive terminals were found in laminae I, II and III. Some of these GABA- and glycine-like immunoreactive terminals were observed chiefly to make symmetric synapses with HRP-labeled neuronal cell bodies and dendritic processes. The present results indicate that neurons in the medullary dorsal horn projecting to the lateral parabrachial region might be modulated by GABAergic and glycinergic inhibitory intrinsic neurons, which might be significantly involved in the regulation of the noxious information transmission.

Fos expression in serotonergic midbrain neurons projecting to the paraventricular nucleus of hypothalamus after noxious stimulation of the stomach: a triple labeling study in the rat.

Horseradish peroxidase (HRP) was stereotaxically injected into the paraventricular nucleus of the hypothalamus (PVH) and visceral noxious stimulation was given by an administration of formalin into the stomach. The brain sections were subjected to HRP histochemistry and immunostained with Fos and serotonin antibodies. The periaqueductal gray (PAG) and dorsal raphe nucleus (DR) contained single-labeled (Fos, 5HT, HRP), double-labeled (Fos/HRP, Fos/5HT, HRP/5HT) and triple-labeled (HRP/Fos/5HT) neurons. Triple-labeled neurons were mainly observed in the DR and in discrete areas of the PAG ipsilateral to side of the HRP injection. These findings suggest that a population of serotonergic midbrain neurons receive nociceptive visceral information and project to the PVH.

Medullary catecholaminergic neurons projecting to lateral hypothalamic area and expressing Fos after chemical stimulation of the stomach in the rat.

Catecholaminergic neurons in the medulla projecting to lateral hypothalamic area (LH) and expressing Fos were investigated in the rat by a triple labelling method, in which horseradish peroxidase (HRP) was injected into the LH, and visceral noxious stimulation was induced by formalin injection into the stomach, sections of the medulla were stained histochemically for HRP and immunohistochemically for Fos and tyrosine hydroxylase (TH). Some neurons labeled with HRP and showing both Fos- and TH-like immunoreactivities were mainly found in the nucleus tractus solitarii (NTS) and ventrolateral medulla (VLM) at the middle and caudal levels of medulla, only a few of them were located in the reticular formation between NTS and VLM. These results indicated that some medullary catecholaminergic neurons projected to the LH and some of them might be involved in the transmissing processes of stress responses to the visceral nociceptive information produced by chemical stimulation.

Fos expression in catecholaminergic medullary neurons induced by chemical stimulation of stomach projecting to the paraventricular nucleus of the hypothalamus in rats.

AIM: To determine whether medullary catecholaminergic neurons expressing Fos induced by chemical stimulation of the stomach project to the paraventricular nucleus of hypothalamus (PVH) in rats. METHODS: Horseradish peroxidase (HRP) was introduced stereotaxically into the PVH of rats. Histochemical analysis of coronal sections through the medulla were analyzed using triple-label immunohistochemistry to identify cells that were retrogradely labeled with HRP, Fos (ABC method), and tyrosin hydroxylase (TH) (PAP method). RESULTS: Seven kinds of labeled neurons were found in the nucleus tractus solitarii (NTS), the ventrolateral medulla (VLM) and the reticular formation (RF) of the medulla: Fos-like immunoreactive (FosLI) neurons, TH-like immunoreactive (TH-LI) neurons and HRP retrogradely single-labeled neurons, FosLI/HRP, FosLI/TH-LI and HRP/TH-LI double-labeled neurons, and FosLI/HRP/TH-LI triple-labeled neurons. CONCLUSION: Ascending projections from the NTS, VLM and RF to the PVH might be involved in the transmitting process of visceral noxious stimulation.

[Synthesis and biological activity of substance P analogs].

Substance P (SP) and its three analogs were synthesized through solid-phase procedure. The fully-protected peptide-resin was cleaved with anhydrous HF.--SH of Cys in [Cys5,9] SP(4-11) was protected with Acm, then deprotected through Iodine oxidation method, the disulfide bridge cyclization was formed subsequently. The crude product was purified on C18 RP-HPLC. The results of guinea-pig isolated ileum test (GPI) showed that the potency of the analogs was as follows: [AcGln5, Pro9] SP(5-11) > SP > SP(4-11) > [Cys5,9] SP(4-11). They induced GPI to contract very fast and showed the characteristics of tachykinin. On the writing test in mice, the results showed: [Cys5,9] SP(4-11) > SP > SP(4-11) > [AcGln5, Pro9] SP(5-11). The biological tests showed that the SP analogs have some extent of selectivity.

[Synthesis and biological activity of dynorphin A-(1-13) analogs].

Dynorphin A-(1-13) (I) and its two analogs were synthesized by solid-phase method. Fully-protected peptides were cleaved from the resin with HF. The three products were purified and their analgesic activity (antiwrithing response in mouse) and receptor affinity in mouse vas deferens (MVD) and rabbit vas deferens (RVD) were measured. The biological and pharmacological results showed that the replacement of Gla2, Ile8 and Pro10 by D-Ala8 and D-Pro10 caused an increase in analgesic activity, receptor affinity and selectivity. In analgesia and RVD assay, its activity is 2.6 times and 135 times respectively higher than dynorphin A-(1-13). The structure-activity relationships were briefly discussed.

Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons projecting from the periaqueductal gray to the ventromedial hypothalamic nucleus in the rat.

Direct projections from serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) to the ventromedial hypothalamic nucleus (VMH) in the rat were investigated by the retrograde horseradish peroxidase tracing method combined with the immunocytochemical technique. Serotonin-, substance P- and tyrosine hydroxylase-like immunoreactive PAG neurons sending their axons to the VMH were distributed in the ventrolateral subnucleus and ventral portion of the medial subnucleus of PAG at the middle and caudal levels.

[Comparison of pharmacological profile of selective kappa-opioid agonist K-II and U-50488].

K-II is an analogue of U-50488, a selective kappa-opioid agonist. Comparison of pharmacological profile of K-II and U-50488 was studied using in vitro and in vivo methods. The results showed that the IC50 values of K-II and U-50488 on electrically induced contraction of the rabbit vas deferens were 0.42 nmol/L and 26.5 nmol/L, respectively. ED50 values of K-II and U-50488 in impairing motor function of mouse in the horizontal screen test were 1.7 and 15.3 mg/kg, respectively. The effect of K-II in reducing spontaneous activity of mouse was 6 times as potent as U-50488. These results suggest that K-II is a more potent kappa agonist in pharmacological effects than U-50488.