Objective To develop a rapid and reliable method for determination of 210Po using large-area grid ionization chamber α spectrometry.Methods Samples were digested using a microwave digestion system.After preparation of sample source,the concentration of 210Po in clam was detected by large-area grid ionization chamber (φ 25 cm).209Po tracer was used to obtain the recovery.Results Large-area grid ionization chamber could achieve better counting and α spectrum resolution when the optimized thickness was 250 μg/cm2.By spiking 209Po tracer in clam,the minimum detectable activity was 9.870 × 10 4 Bq and the recovery of 210Po was 98％.Conclusions Compared with the traditional method,the developed method can avoid separation process,using less quantity of sample (0.2-0.5 g dry) and simplify the measurement process.This method may be has broad application prospects.

Background The fate of adult stem cells is associated with its surrounding microenviroment.Our previous work found that embryonic stem cells (ESCs) micro-environment enhance the stemness of human limbal stem cells (LSCs),but its mechanism has not been elucidated.Objective This study was to explore the molecular mechanism of ESC micro-environment enhancing the stemness and inhibiting the apoptosis of LSCs.Methods Human LSCs were cultured by explant culture method with CnT-20 medium and CnT-20+20％ ES culture supernatant (ESC-CM),respectively.Colony formation assay was used to analyze the proliferation ability of cells.Telomerase reverse transcriptase (TERT) siRNA (19-25nt siRNA) or siRNA (sc-37007) was transfected into the cells of ESCCM group.Apoptosis and mitochondrial membrane potential were assayed by flow cytometry,and the expressions of telomerase and reactive oxygen species (ROS) in TERT siRNA-or siRNA-F-transfected cells by immunofluorescence and flow cytomery.RT-PCR,immunofluorescence staining and Western blot were employed to determine the expressions of p63,ATP-binding cassette transporer G2 (ABCG2),integrin β1 mRNA and proteins and cytokeratin 3 (C K3) in the cells.The levels of focal adhesion kinase (FAK),Akt,glycogen synthase kinase 3β (GSK3β) and p21 protein and phosphorylation proteins in the cells were detected by Western blot.Results The LSCs presented an increased proliferative capacity and passaged to the eighth generation with the colony-forming efficiency (CFE) of (7.6±0.6) ％ in ESC-CM group,but the cells to the sixth generation with the CFE of (5.6±0.6)％,showing a significant difference between them (t =4.454,P =0.011).The apoptotic rates of the cells from 2 through 6 generations were lower in the ESC-CM group than those in the CnT-20 group (all at P＜0.05).The apoptotic rate of the cells was (7.67± 1.31)％ in the siRNA-F transfected group,which was significantly lower than (32.33 ±3.13)％in the siRNA-TERT transfected group (t =-12.588,P =0.000).No significant differences were seen in the expression levels of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the primary cells between the ESC-CM group and the CnT-20 group (all at P＞0.05),but significantly declined expressions of CK3 mRNA and protein were found in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).However,the expressions of p63,ABCG2,integrin β1 mRNA and proteins and TERT protein in the second generation of the cells were significantly higher in the ESC-CM group compared with the CnT-20 group (all at P＜0.01).The telomerase activity was (4.83±0.67) ％ in the siRNA-TERT transfected group,which was significantly lower than (46.71±1.22) ％ of the siRNA-F transfected group (t =52.116,P =0.000).The expression of pFAK,pAkt,pGSK3β proteins were weakened,but the expression of p21 was increased in the ESC-CM group after addition of FAK inhibitor,GSK3β inhibitor and TERT-siRNA transfected group.Mitochondrial membrane potential in the second generation of cells was elevated in the ESC-CM group in comparison with the CnT-20 group and the siRNA-TERT transfected group (all at P＜0.01),and the rates of ROS positively reaction was lower in the ESC-CM group and the siRNA-F transfected group than those of the CnT-20 group and siRNA-TERT transfected group (all at P＜0.01).Conclusions ESC-CM culture system can effectively keep the stemness of LSCs and inhibit apoptosis.ESC-CM culture system plays functions probably via telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways.

Objective To investigate the predictive value of abnormal multiples of the median (MoM) of second trimester maternal serum triple screening (STMSTS) markers for adverse pregnancy outcomes.Methods 16 000 singleton pregnancies at 15+0 to 20+6 weeks' gestation who underwent STMSTS between July 2010 and January 2013 in the First Hospital of Jilin University were recruited.Maternal serum AFP,free β-hCG (F-β-hCG) and unconjugated estriol (uE3) levels were measured using time-resolved fluoroimmunoassay,and then convened to MoM.LifeCycle 3.2 software was used to calculate risk,and a risk value greater than 1 in 270 or 1 in 350 was considered as high risk for trisomy 21 syndrome (Down syndrome,DS) and trisomy 18 syndrome (Edwards syndrome,ES),respectively.MoM of AFP more than 2.5was considered high risk for open neural tube defect (ONTD).Amniocentesis and karyotyping,ultrasound screening were advised for high risk women.AFP,F-β-hCG higher than 2.0 MoM or uE3 lower than 0.5MoM was considered as abnormal,respectively.The MoM of STMSTS marker between women with adverse pregnancy outcome and with normal outcome was compared.Results (1) The median MoM of AFP,F-β-hCG and uE3 was 0.91 MoM,0.94 MoM and 1.05 MoM,respectively.Of the 16 000 pregnant women,there was no statistical difference in the median MoM of triple screening marker at different weeks of gestation (P＞0.05).The positive rate of DS,ES and ONTD in women ≤35 years old (n=14 972) was 4.03％ (603/14 972),0.36％(54/14 972) and 0.29％(44/14 972) respectively.And in women＞35 years old(n=1 028),the positive rate was 24.51％ (252/1 028),1.95％ (20/1 028) and 0.78％ (8/1 028),respectively.There was a statistically significant difference of positive rate between the two groups(P＜0.05).(2) 9 cases of DS,1 case of ES and 1 ease of ONTD were found in the high risk group,and 2 cases of DS in the low risk group.The detection rate of DS,ES and ONTD was 9/11,1/1 and 1/1 respectively; and the positive predictive value was 1.05％(9/855),1.35％(1/74) and 1.92％(1/52),respectively.(3)The incidence of adverse outcome (group 1) was 1.49 ％(239/16 000).7 760 pregnant women in this study were healthy during pregnancy,so were their fetuses (group 2).There were significant differences in the age at delivery,body weight and markers' MoM of STMSTS between the two groups(P＜0.01).(4) In group 1,the rate of abnormal MoM of AFP or F-β-hCG was 7.95％(19/239) and 23.85％ (57/239),and the abnormal rate of MoM of uE3 was 4.18％(10/239).The rate of two abnormal MoM of markers was 5.02％(12/239); the rate that all three MoM were abnormal was 0.84％(2/ 239).However,in group 2,the rate of two abnormal MoM of markers was 0.14 ％(11/7 760); and the rate that all three MoM were abnormal was 0.There was a significant difference of abnormal MoM of maternal serum marker between the two groups (P＜0.01).Conclusions There is a relationship between abnormal marker of STMSTS and adverse outcomes.STMSTS show a high value in the detection of DS,ES and ONTD.

Background Recent studies indicated that human amnion mesenchymal stem cells (hAMSCs) can be induced to differentiate into multiple types of cells in vitro,but whether the hAMSCs can differentiate into ocular surface cells has not been reported yet.Objective This study was to investigate the feasibility of inducing differentiation of hAMSCs into ocular surface cells by co-culturing with human bulbar conjunctiva fibroblasts (hBCFs).Methods This study was approved by Ethic Committee of Affiliated Second Hospital of Guangzhou Medical University.HAMSCs were isolated from placenta under the informed consent of healthy delivery women.hAMSCs were cultured,passaged and identified by detecting the expressions of CD44,CD45,CD73,CD90 in the cells with flow cytometer,osteogenesis and adipogenic differentiation experiments.Human conjunctival tissue was obtained during the eye operation under the informed consent of patients and hBCFs were isolated and cultured with explant culture.The cells were divided into the hAMSCs culture group and the hAMSCs and hBCFs co-culture group and cultivated in Transwell chambers for 7 days.The expressions of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in the cells were assayed by immnofluorescence technique.Results Cultured hAMSCs showed the slender shape and cell body enlarged with passage.CD44,CD73 and CD90 were expressed in the cells,and the expression of CD45 was absent.After 3-4 weeks of osteogenesis and adipogenic induce,the cells showed red staining for alizarin and oil red O.In the co-culture group of hAMSCs and hBCFs,hAMSCs presented the epithelioid cell-like in shape and showed the positive response for CK19 and weaker response for α-SMA.However,in the hAMSCs culture group,the cells showed the positive response for α-SMA and absent response for CK19.Conclusions The hAMSCs can differentiate into ocular surface cells after being induced by hBCFs.And the differentiation mechanism is possibly relevant to mesenchymal cells epithelium.

Objective To study the influences of the length of sexual abstinence on sperm parameters and seminal plasma biochemical indicator.Methods Sperm concentration and motility were detected by computer assisted sperm analysis(CASA).The sperm morphology was analyzed using semiautomatic sperm morphology analyzer.Seminal plasma fructose concentration,neutral a-glycosidase,zinc,and acid phosphates concentration were measured by spectrophotometer.Improved DTNB was conducted for detection of the content of seminal plasma novain.Prostatespecific antigen(PSA)was measured with commercial kit.Three groups were defined according to the days of sexual abstinence:G1(1-3 days),G2(4-5 days)and G3(＞6 days).Results Sperm concentration of G2 was significantly higher than that of G1(P＜0.01),and of G3 than that of G1 and G2(P＜0.01).Sperm motility and per centage of morphologically normal sperm of G3 were significantly lower than that of G1(P＜0.01).Seminal plasma fructose concentration of G3 was significantly lower than that of G1 and G2(P＜0.01,P＜0.05).Seminal plasma neutral a-glycosidase,carnutine,zinc concentration of G3 was significantly higher than that of G1 and G2(P＜0.01,P＜0.05).Conclusion The length of sexual abstinence can influence sperm parameters and seminal plasma biochemical indicator.

Three fresh lungs removed from three male adult dogs were injected with ABS. This material was injected into each lobe of the lung through the pulmonary artery and bronchus simultaneously. The pressure was maintained between 200～280 mmHg. The injected specimens were digested in 20～50％ HCl and peptic solutions for 7～10 days. A part of the cast replicas of the lungs was taken off and gilded with EIKO IB-3. Under SEM (HITACHIS～450) the specimens thus prepared were observed. The chief findings were as follows:Scanning electron micrographs of the injected vascular system presented a clear, three-dimensional picture of extensive capillary networks around the individual alveoli. In the septa between alveoli the capillary networks appeared to be a single layer. These networks looked like pentagonal or hexagonal rings; the meshes in between them were smaller than the vessels themselves.The capillaries observed here in the scanning stereopicture belonged to the category of the alvioli cappilary of the subpleura; the meshes of which were larger than those of other parts.Because the numerous alveoli and capillary networks surrounding the alveoli were filled with the ABS, the position and the shape of the capillary networks can be seen very clearly.The lumen of the alveoli were polyhedron in shape, various in sizes and smooth in contour. Many imprints of the alveolar type Ⅱ cell nuclei existed on the surface. The bridge formation between alveoli were really interalveolar pores; their number and diameter varied. The function of the interalveolar pores was briefly considered in this paper.

The present study intended to provide some informations about the relationship between the supraependymal structures (SES) and the periventrieular neural tissue (PVNT), and with gross dissection and scanning electron microscopy the anterior medullary velum (AMV) was observed on 20 adult rabbits. The AMV may be divided into three portions: 1. the posterior membraneous wall of the recess of the inferior colliculus; 2. the anterior roof of the fourth ventricle; and 3. the transiional portion between the two portions mentioned above. On the ependymal surface of the entire AMV. There were numerous microvilli and cilia, except for the second portion of AMV, there were also some spherical-like structures, which were 2-6?m in diameter and had lace-like processes on its surface, and supraependymal cells(SEC) which were stellate, triangular, and spindle in shape. On the cellular surface with very few SEC, the secretory granules may be seen, which were 0.1-0.3?m in diameter. The SEC often extended out 2-5 processes, and the distal parts of which expanded into a flattened shovel-like structure, which lay on or inserted into the ependymal surface. The SEC here are similar to type Ⅱ SEC seen in the third and fourth ventricles, but they may differ significantly in structures on cellular surface, e.g. the secretory granules, and in shapes of their processes. Thus, it may suggest that the SEC here may play an intermediary role between PVNT and CSF, and be another route of neurohumoral modulation.

A 14-week human fetal kidney was studied by the method of freeze-fracture, the results were as follows:1. All the nuclei of various cells at this stage of development possess distinct nuclear pores with the same diameter and they are distributed randomly on the nuclear membrane. The intramembraneous particles on the PF of both the inner and outer nuclear membranes are more numerous than that of the EF. The morphological features of the nuclear pores vary according to the plane of their fracture face; they appear as dimples on the EF and as valcano mouth on the PF. The interior of the nucleus usually contains homogeneously dispersed particles, but no such structures were seen in thin sections. In some nuclei a round vesicle was revealed.2. The cell membrane of various cells, at this stage, shows special structures to manifest their degree of differentiation. In the less differentiated epithelial cells of the renal tubules, the cell membrane is straight and the intramembraneous particles are randomly distributed, the intromembraneous particles on PF are more than on EF, comparetively well developed tight junctions are located at the latexal surface near the apical portion of the cell while in the differentiated proximal tubules, there are many microvilli on their luminal surface, but their lateral cell membrane is still straight without interdigitations. In some tubules small processes near the basal part may be seen, however, basal fold is still absent. The basal membrane of the renal tubules is very prominent. As to the epithelial cells of the viseral layer of the renal corpuscle, gap and tight junctions are revealed while they are columnar in shape, but as they differentiated into podocyte with primary and secondary processes, the structure of tight junctions become simple, less prominent and discontinuous.3. Cytoplasm: In the process of cell differentiation the amount of cytoplasm increases and the ratio of nuclear end cytoplasmic volume decreases. The orgauelles are less in the undifferentiated cells, so the structure of the cytoplasm of them is much simpler than that of the more differentiated ones. The compartmentation phenomenon of the cytoplasm of differentiated cells become distince. In the freeze-fracture micrographs the membraneous structures, such as mitochondria, Golgi complex and secretion granules etc. are more stereoscopic than those in thin sections, but the opportunity to reveal the fine structures of various cells is minimized due to only one fracture face for each specimen and what structure will be fractured is beyond the control of the operator.

The livers from 5 week 14～32 human fetuses were studied. The results are summarized as follows:1. Two kinds of liver cells revealed by EM: the "light" and "dark" cells. The light ones are larger in size, more numerous in number, containing larger mitochondria and obvious RER and SER, but less free ribosomes than the dark ones. Both of them may contain hemosiderin granules and the configuration of mitochondria enclosed by circular or semicircular RER cisternae.2. Bile canaliculus: Beside canaliculi between two adjacent liver cells, canaliculi situated between 3 or 4 liver cells are not infrequently revealed. That is due to the fetal liver cells are arranged in groups rather than in cords. The stereoscopic configurations of bile canaliculus and the tight junction surrounding it are demonstrated more clearly in freeze-fracture micrograph than that of the TEM. In addition, one intracellular bile canaliculus is found in the freeze-fracture preperation.3. The blood sinusoid and hemopoietic focus: In the 4-week fetus, the endothelium of the sinusoid can hardly be recognized While its lumen is quite large. Many erythrocytes accompanied by their immature components can be seen among it. As to the 32-week fetal liver, the endothelium and Kupffer cell may be easily recognized but the lumen of the sinusoid dimenishes enormously. The hemopoietic foci are localized extra-sinusoidially and in close contact with liver cells. Their volume exceeds one half of the fetal liver lobule before the age of 28 weeks, then it dimenishes gradually. In the space of Disse a fat storage cell is revealed.4. The portal canal : The mensenchyma and immature erythrocytes in the portal canal dimenishes with the increase of the fetal age. The portal canal is surrounded by the pigmented limiting plate cells of the liver lobules. The interlobular bile duct is differentiated from the limiting plate cells. Its epithelium is composed of both light and dark cells. The former is more numerous and contains many large mitochondria in its apical portion, while the latter shows higher electron density. Microvilli are located on the surfaces of both kinds of cells and finger like processes in the enlarged intercellular spaces are revealed.

In order to elucidate the ultrastructural changes of human spermatozoa afteradministration of gossypol acetate 40 mg/day for 50 days,freeze-replica techniqueswere employed.In addition to the ordinary changes of spermatozoa,such as swelling,wasting,atrophy,tortuosity,nodulation in the head and/or tail region and fracture betweenhead and tail,the internal structure,biomembrane and intramembranous particles ofsperm also showed relatively more sensitive changes.The changes of biomembranerevealed certain shift in regard to the firmness of reciprocating attachment of inner-and outer-leaflets of the unit membrane.On the other hand,the changes of intra-membranous particles revealed a specialized arrangement,this is,on the plasmamembrane of head and tail they were concentrated into lattice work,whereas,in thehind-head region they accumulated as clusters.In the hind-acrosomal membranetwo types of particles,large and small,may be distinguished,the smaller ones werearranged orderly in lines of all directions.As for the nuclear pores,no changewas found in this study.It is considered that the influences of gossypol upon human spermatozoa,firstof all,were shown in the biomembrane and intramembranous particles,and theordinary changes in appearance were the secondary result.In the discussion,it'ssuggested that a special agent,gossypol receptor,responsible for the gossypol effectsto the sperm,may be present in the plasma membrane of the spermatozoa.