Establishment and Evaluation of Intestinal Injury Model of Mouse Acute Graft Versus Host Disease Based on An Organoid Technology / 中国实验血液学杂志

OBJECTIVE@#To establish an intestinal organoid model that mimic acute graft versus host disease (aGVHD) caused intestinal injuries by using aGVHD murine model serum and organoid culture system, and explore the changes of aGVHD intestine in vitro by advantage of organoid technology.@*METHODS@#20-22 g female C57BL/6 mice and 20-22 g female BALB/c mice were used as donors and recipients for bone marrow transplantation, respectively. Within 4-6 h after receiving a lethal dose (8.0 Gy) of γ ray total body irradiation, a total of 0.25 ml of murine derived bone marrow cells (1×107/mice, n=20) and spleen nucleated cells (5×106/mice, n=20) was infused to establish a mouse model of aGVHD (n=20). The aGVHD mice were anesthetized at the 7th day after transplantation, and the veinal blood was harvested by removing the eyeballs, and the serum was collected by centrifugation. The small intestinal crypts of healthy C57BL/6 mice were harvested and cultivated in 3D culture system that maintaining the growth and proliferation of intestinal stem cells in vitro. In our experiment, 5%, 10%, 20% proportions of aGVHD serum were respectively added into the organoid culture system for 3 days. The formation of small intestinal organoids were observed under an inverted microscope and the morphological characteristics of intestinal organoids in each groups were analyzed. For further evaluation, the aGVHD intestinal organoids were harvested and their pathological changes were observed. Combined with HE staining, intestinal organ morphology evaluation was performed. Combined with Alcian Blue staining, the secretion function of aGVHD intestinal organoids was observed. The distribution and changes of Lgr5+ and Clu+ intestinal stem cells in intestinal organoids were analyzed under the conditions of 5%, 10% and 20% serum concentrations by immunohistochemical stainings.@*RESULTS@#The results of HE staining showed that the integrity of intestinal organoids in the 5% concentration serum group was better than that in the 10% and 20% groups. The 5% concentration serum group showed the highest number of organoids, the highest germination rate and the lowest pathological score among experimental groups, while the 20% group exhibited severe morphological destruction and almost no germination was observed, and the pathological score was the highest among all groups(t=3.668, 4.334,5.309,P<0.05). The results of Alican blue staining showed that the secretion function of intestinal organoids in serum culture of aGVHD in the 20% group was weaker than that of the 5% group and 10% of the organoids, and there was almost no goblet cells, and mucus was stainned in the 20% aGVHD serum group. The immunohistochemical results showed that the number of Lgr5+ cells of intestinal organoids in the 5% group was more than that of the intestinal organoids in the 10% aGVHD serum group and 20% aGVHD serum group. Almost no Clu+ cells were observed in the 5% group. The Lgr5+ cells in the 20% group were seriously injuried and can not be observed. The proportion of Clu+ cells in the 20% group significantly increased.@*CONCLUSION@#The concentration of aGVHD serum in the culture system can affect the number and secretion function of intestinal organoids as well as the number of intestinal stem cells in organoids. The higher the serum concentration, the greater the risk of organoid injury, which reveal the characteristics of the formation and functional change of aGVHD intestinal organoids, and provide a novel tool for the study of intestinal injury in aGVHD.

Effects of Long-term Sleep-deprivation on Rats′Learning and Memory Behavior and Neurotransmitters in Hippocampus and the Effects of Prevention of Electro-acupuncture on the Sishencong Acupoints / 中山大学学报(医学科学版)

[Objective]To investigate the effects of continuous long-term sleep deprivation on rat's behavior,memory and neurotransmitters in hippocampus,as well as determine the effects of prevention of electro-acupuncture.[Method]We used multi-platform water environment to make the rat model of insomnia.The experiment was divided into two sections.Sec-tion one:thirty rats were divided into 5 groups(six rats each group):tank control group(TC),sleep-deprived for continu-ous 7 days group(SD group),three days of recovery after SD group(3d after SD group),six days of recovery after SD group (6d after SD group),eleven days of recovery after SD group(11d after SD group).The term of recovery refers to normal feed-ing and sleeping when rats return to cages after sleep-deprivation.The open-field test was performed following the end time of each group experiment and then the rats were perfused and fixed.Section two:Eighteen rats were divided into three groups (six rats each group):tank control group(TC),sleep-deprived for continuous 7 days group(SD group),sleep-deprived rats treated with electro-acupuncture group(SD+EA group).Rats of SD group and SD+EA group were deprived sleep for con-tinuous 7 days. During the period of sleep deprivation,the rats of SD+EA group were treated with electro-acupuncture 20 minutes every morning.At the end time of experiment,rats were tested by the Morris water maze and then perfused and get specimens.[Results]Section one:the statistical analysis of outcome of open-field test showed:as compared to the TC group,the scores of rat's horizontal movement and vertical movement were significantly fewer in the SD group(P<0.05).Af-ter 3,6,or 11 days recovery of sleep,the scores increased significantly as compared with SD group(P<0.05). Although, the outcome of open-field test in the 3d after SD and 6d after SD groups had the significant difference with TC group(P<0.05),there was no different between the 11d after SD group and TC group(P>0.05).The quantitative analysis of gamma aminobutyric acid(GABA)expression in the hippocampus showed:the SD group shows lower level of GABA when com-pared to the TC group(P<0.05).After 3 days or 6 days of recovery,the expression of GABA is higher than the SD group(P<0.05),and similar to the TC group(P>0.05).In the 11days after SD group,the expression of GABA declined as compared to the TC group and the 6d after SD group(P<0.05).Section two:In the Morris water maze experiment,when compared to the TC group and the SD+EA group,the escape latency period was significantly prolonged and the times of across the plat-form were significantly reduced in the SD group(P<0.05).On the contrary,there were no statistical differences in the escape latency period or the across platform times between the SD+EA group and the TC group(P>0.05).The results of serotonin(5-HT)and GABA in the hippocampus showed:the 5-HT level in the SD group was no significant difference as compared to the TC group and the SD group on 5-HT level,while the 5-HT level in the SD+EA group was reduced as compared to the TC group(P<0.05). In the opposite,the GABA level in the SD group was decreased as compared to the TC group(P<0.05),while the SD+EA group and the TC group appear similarly level(P>0.05).[Conclusions]The effect of 7 days'contin-uous long-term sleep-deprivation may lead to the reduction in the ability of learning and memory behavior of rats.Our results is different with 4 days'continuously sleep-deprivation which was reported on the rats'expression of GABA and 5-HT. Electro-acupuncture stimulating the Sishengcong Acupoints seems preventing the harming of long-term sleep-deprivation on the ability of learning and memory,which probably is through adjusting the disorder of central neurotransmitter expression caused by sleep deprivation.

Effects of Rhodiola on the Expression of iNOS mRNA in Severe Acute Pancreatitis Associated Re- nal Injury Rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the effect of Rhodiola on the expression of iNOS mRNA in severe acute pancreatitis (SAP) associated renal injury rats.</p><p><b>METHODS</b>A total of 72 healthy rats were randomly divided into the sham-operated group (S), the SAP associated renal injury group (M), and the Rhodiola-treated group (RHO), 24 in each group. Rats in S and M groups were peritoneally injected with 10 mL/kg saline 3h before modeling, while rats in the RHO group were peritoneally injected with 10 mL/kg Rhodiola Injection 3 h before modeling. The peripheral ligament of pancreas was bluntly dissociated in rats of M and RHO groups. The head of pancreas was occlused by nontraumatic blood vessel forceps 3 h later to establish the model. Eight rats were randomly selected from each group at 12, 24, and 36 h after modeling to detect levels of serum amylase, creatinine, and blood urea nitrogen. Serum levels of interleukin 1β (IL-1β) and interleukin 10 (IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). Pathological changes of the left kidney were observed under light microscope. The expression of inducible nitric oxide synthase (iNOS) mRNA in the right kidney was detected with real time polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>Compared with the S group, serum levels of amylase, creatinine (Cr), blood urea nitrogen (BUN), IL-1β, IL-10, and iNOS mRNA expression significantly increased in the M group (P < 0.01). The function of kidney and pancreas were obviously improved in the RHO group than in the M group. Levels of IL-1β and iNOS significantly decreased, but IL-10 levels significantly increased in the RHO group with statistical difference (P < 0.05).</p><p><b>CONCLUSION</b>Rhodiola had better protective effect on SAP associated renal injury, which might be achieved through inhibiting the expression of IL-1β, stimulating the expression of IL-10, down-regulating iNOS mRNA expression, reducing the generation of oxygen free radicals and NO damage to cells, and improving hypoxia tolerance capabilities of the kidney.</p>

ATM/H2AX and repair of sperm-DNA damage during cryopreservation / 中华男科学杂志

Semen cryopreservation is an important method in assisted reproductive technology, but meanwhile it is complicated by cryodamage to spermatozoa. Mechanisms behind the cryodamage to spermatozoa are thought to be multifactorial, and the excessive generation of reactive oxygen species (ROS) has been suggested as a major contributing factor. DNA-damaged sperm is also capable of fertilizing oocytes, significantly affecting the outcome of reproduction. As a sensor of DNA damage responses, protein kinase ATM (ataxia telangiectasia mutated) can be initiated through rapid intermolecular autophosphorylation induced by DNA damage, phosphorylate various proteins, and amplify the responses to DNA damage. After DNA damage, histone H2AX is activated by ATM, which contributes to the repair of sperm-DNA damage after fertilization and regulates the cell cycle during embryo development. Given the important role of the ATM/H2AX signaling pathway in the response to and repair of DNA damage induced by oxidative stress, it may mediate the repair of sperm-DNA damage resulting from cryopreservation.

Genetic analysis and serum level of cartilage oligomeric matrix protein in patients with pseudoachondroplasia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pseudoachondroplasia (PSACH) is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein (COMP). Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation. There is evidence that decreased serum COMP concentration may serve as a diagnostic marker in PSACH. Here, we investigated the role of this gene and the serum COMP concentration in Chinese patients with PSACH.</p><p><b>METHODS</b>A family with three patients and a sporadic case were recruited. Genomic and phenotypic data were recorded. The diagnosis of PSACH was made on the base of clinical evaluation. The genomic DNA was extracted from peripheral blood leukocytes. The 8-19 exons and flanking intron-exon boundary sequences of COMP were amplified by polymerase chain reaction (PCR) and screened for mutation by direct DNA sequencing. Serum COMP concentrations of 4 patients and age-compatible control group of 20 unrelated healthy subjects were analyzed on the basis of an ELISA Kit for human cartilage oligomeric matrix protein.</p><p><b>RESULTS</b>A deletion (c.1447-1455del) was identified in exon 13 in the sporadic case. The mean serum COMP concentrations of four patients (3.12+/-2.28) were significantly lower than those of control group (10.86+/-2.21, P<0.05). There was no overlap in the distribution of serum COMP concentration between PSACH patients and controls.</p><p><b>CONCLUSIONS</b>Mutations in COMP gene are responsible for the PSACH. Serum COMP concentration may be suggested as an additional diagnostic marker to aid clinical findings in suspected cases of PSACH.</p>

GammaH2AX-mediated repair of DNA damaged sperm in the zygote / 中华男科学杂志

Male germ cells are particularly susceptible to DNA damage by genotoxic agents during spermiogenesis and spermatozoal maturation, and meanwhile lack an effective repair system to eliminate the lesions. Because the DNA damaged sperm still has fertilizability and developmental potentiality, damage repair may occur after fertilization, but its mechanism remains unknown. Histone H2AX phosphorylation (gammaH2AX) is reportedly involved in the repair of damaged sperm DNA after fertilization. This review aims to summarize the present knowledge on the mechanism of gammaH2AX-mediated repair of DNA damaged sperm in the zygote.

Effects of exposure to estradiol valerate in early pregnancy on the reproductive system development of F1 male rats / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish the rat model of estradiol valerate medication in early pregnancy, and to investigate the effects of estradiol valerate on the development of the reproductive system of the first filial generation (F1) male rats by evaluating the anogenital distance (AGD) and the development of the testis and epididymis.</p><p><b>METHODS</b>Pregnant SD rats were divided at random into a blank control group and a low dose, a medium dose and a high dose medication group to receive intragastric estradiol valerate at 0.2 mg/kg, 0.5 mg/kg and 0.8 mg/kg, respectively. The newborn F1 male rats were normally fed. Their anogenital distances were measured on postnatal day (PND) 3 and 21, the organ coefficients of the testis and epididymis (testicular and epididymal weight g/body weight 100 g) were obtained on PND 60, the morphological changes of spermatogenic cells were observed by testis biopsy, and the diameter of the seminiferous tubule and epithelial height were measured.</p><p><b>RESULTS</b>There was no significant difference between the control and medicated F1 male rats in AGD on PND 3 and 21 (P > 0.05), nor in the organ coefficients of the testis and epididymis on PND 60 (P > 0.05), nor in the diameter of the seminiferous tubule and epithelial height.</p><p><b>CONCLUSION</b>Medication of estradiol valerate (0.2 -0.8 mg/kg) to rats in early pregnancy neither significantly affects the reproductive system development, nor induces obvious histological changes of the testis in the sexual maturation period of their F1 males.</p>

Relevant factors of microalbuminuria in aged patients with essential hypertension / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship between microalbuminuria and endothelial-dependent relaxing function and atherosclerosis of common carotid artery (CCA) in aged patients with essential hypertension (EH).@*METHODS@#Sixty-four aged EH patients were recruited. According to the albumin excretion rate (AER) in the urine measured by immunoturbidimetry, patients were divided into 2 groups: normoalbuminuria group (NAU group) and microalbuminuria group (MAU group). Thirty aged persons without EH were served as the control group. The endothelium-dependent relaxing function of blood vessels, intima-media thickness (IMT) and the plaque of CCA were measured by color Doppler ultrasound.@*RESULTS@#The flow-mediated dilation in the MAU group [(4.98+/-1.35)%] and that in the NAU group [(6.31+/-1.14)%] were significantly lower than that in the control group [(9.09+/-1.83)%, P<0.05, respectively], especially lower in the MAU group. The IMT of CCA in the MAU group [(0.97+/-0.19)mm] and that in the NAU group [(0.86+/-0.10)mm] were significantly thicker than that in the control group [(0.78+/-0.13)mm] (P<0.05, respectively), especially thicker in the MAU group. The analysis of multiple stepwise regression showed that the microalbuminuria was successively related to EDF, the IMT of CCA, the plaque index of CCA, systolic blood pressure, etc.@*CONCLUSION@#EDF is impaired, and there is the atherosclerosis of CCA in aged patients with EH. Microalbuminuria correlates with the decrease of endothelium-dependent relaxing function and the IMT of CCA in aged patients with EH.

Intracytoplasmic sperm injection in cases with history of in vitro fertilization failure / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To evaluate the effect of intracytoplasmic sperm injection (ICSI) in the management of cases with a history of conventional in vitro fertilization (IVF) failure.</p><p><b>METHODS</b>Two groups of patients, 19 with normal semen parameters and a history of IVF failure (metaphase II oocytes: 0-30 %) and 28 with severe male factor infertility received ICSI technology during the same period. Ovarian stimulation was achieved by conventional procedure. Transvaginal ultrasound-guided oocyte collection was done 35-37 h after human chorionic gonadotrophin (hCG) injection. Only metaphase II oocytes were selected for microinjection.</p><p><b>RESULTS</b>Fertilization was achieved with ICSI in all the patients. The fertilization rate (75.6 % +/-21.1 % vs. 73.9 % +/-19.2 %), cleavage rate (85.1 % +/-19.3 % vs. 82.7 % +/-22.1 %), clinical pregnancy rate per embryo transfer cycle (31.6 % vs. 28.6 %) and implantation rate per embryo (15.3 % vs. 14.4 %) did not differ significantly between the two groups.</p><p><b>CONCLUSION</b>ICSI is a valuable method for couples with a history of IVF failure. These patients may have a similar ICSI result as in severe male infertility.</p>