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Objective:To explore the establishment and application of ovarian cancer organoids.Methods:Fresh ovarian tumor tissues, obtaining from patients underwent surgery in the First Affiliated Hospital of Nanjing Medical University between October 2021 and March 2022, were collected, enzymatic degraded, digested, and embedded into matrigel to establish organoids. A total of 32 ovarian cancer samples were collected. Hematoxylin eosin (HE) staining and immunofluorescence (IF) procedure were used to verify the morphological structure of organoids and their expression of molecular markers. 3D cyto-live or dead assay was used to detecte the live or dead cells in organoids. Carboplatin with a concentration ranging from 5 to 80 μmol/L (5, 10, 20, 40, 80 μmol/L) was added to organoids to calculate the 50% inhibitory concentration (IC 50) in different organoids. Results:(1) Organoids from a total of 32 patients were established, of which 18 cases could be passaged stably in the long term in vitro, while 14 could be passaged in the short time. The average amplification time of long-term passage in vitro was over 3 months, and the longest reached 9 months. (2) In HE staining, significant nuclei atypia and local micropapillary structures were observed in organoids. IF staining revealed that ovarian cancer organoids expressed molecular markers similar to primary tumor tissues, such as Pan cytokeratin (Pan-CK), p53, paired box gene 8 (PAX8), and Wilms tumor gene 1 (WT1). (3) In 3D cyto-live or dead assay, a large number of apoptotic cells were observed inside and around the organoids after added carboplatin. The sensitivity to carboplatin varied in 18 organoids could amplify in the long term, with an average IC 50 of (29.5±15.8) μmol/L. Moreover, IC 50 values of 4 organoids derived from patients received neoadjuvant chemotherapy were much higher than the 14 organoids which did not received neoadjuvant chemotherapy [(48.7±11.3) μmol/L vs (24.0±12.1) μmol/L; t=3.429, P=0.022]. Conclusions:Organoids recapitulate ovarian cancers in vitro and could be stably passaged. Organoids derived from patients received neoadjuvant chemotherapy have higher resistance to carboplatin.
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Objective To optimize the extraction methods of mitochondrial genome DNA(mtDNA)of Oncomelania hupen-sis. Methods The pyrolysis,protein K variable-temperature digestion and high-concentration potassium acetate purification were applied to optimize the high-concentration-salt precipitation method,and then the optimized method was compared with two common extraction methods,the sucrose density gradient centrifugation method and traditional high-concentration-salt pre-cipitation method. The mtDNA samples were identified by using spectrophotometry,agarose gel electrophoresis and the amplifi-cation products of COX1. The nuclear DNA contamination was tested by the amplification products of ITS. Results The concen-tration and yield of the improved method was significantly higher than those of the traditional method(F=3032.65,10185.00, both P<0.01). The mtDNA samples extracted were essentially free of nuclear DNA and protein,meeting PCR,sequence analy-sis and other molecular biology research requirements. Conclusions The improved high-concentration-salt precipitation meth-od for isolating mtDNA is simple,and it has high yield and low cost. The extracted mtDNA can meet relevant analysis require-ments.
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Objective To understand the breeding situation of Acaroid mites in indoor environments of kindergartens in Wuhu City so as to provide the evidence for its prevention and control. Methods From March to June and September to De?cember in 2014 dust samples were collected from 15 kindergartens of 3 ranks every month. Acaroid mites in the samples were isolated identified and counted. Results Totally 360 samples were selected and 169 samples 46.94% had mite infestation. Pyroglyphidae accounted for the most in the population with the breeding rate of 45.00%. Totally 18 504 mites were found and Dermatophagoides farina and Dermatophagoides pteronyssinus were the dominant species with the composition ratios of 30.21%5 590 mites and 17.83% 3 300 mites respectively. The third?ranked kindergartens were in the most serious situation con?cerning with 81.67% 98/120 of the breeding rate of Acaroid mites. The differences were statistically significant among the kin?dergartens with different ranks F=6.048 χ2=73.523 both P0.05 . Conclusion Acaroid mites in the kindergartens in Wuhu City were in serious condition and the population and species composition of Acaroid mite community is influenced by the human behavior and indoors environments.
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Objective To determine the viability of Schistosoma japonicum cercariae by staining. Methods Schistosoma japonicum cercariae were stained by 0.4%trypan blue 0.5%methylene blue?eosin?borax M.E.B 0.5%eosin 0.5%methy?lene blue and 0.05% neutral red respectively for 5 min then they were observed under a stereoscopic microscope. Results The dead cercariae were stained in the trypan blue M.E.B eosin and neutral red but unstained in the methylene blue. The vi?tal cercariae were unstained in all the five kinds of dyes. Conclusion The staining methods by using 0.4% trypan blue 0.5%M.E.B 0.5%eosin and 0.05%neutral red can be used to determine the viability of S. japonicum cercariae.
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Objective To explore the relationship between the Yangtze River floodplain ecological environment(vegetation, soil,water and light intensity)and the distribution of Oncomelania hupensis snails,so as to provide the evidence for ecological snail control. Methods Three regions(the Lu-Gang Bridge,Dragon Nest Lake in the bund,and Dragon Nest lake beach)were selected to investigate the plant characteristics(species,height,coverage,frequency and strain of clusters),soil characteristics (temperature,humidity,light intensity)and pH value. All the results were analyzed statistically with SPSS 18 software. Results A total of 920 boxes were investigated. The vegetation coverage was 3.7%-63.5%,and the dominant population was Cyperusrotun-dus L. cluster on the marshland. The soil temperature was 19.0℃-24.0℃,pH 5.0-5.7,and humidity 53%-75%. There were statis-tical significants in average number of living snails and dead snails among 3 groups( P<0.05). As the light intensity was strong in summer and weak in winter,the snails were in the back surface in summer and front surface in winter respectively. The average number of living snails was the most near the water. The difference was statistical significant(P<0.01)among three ranges(0-1 m,1-3 m and 3-5 m). Conclusion The snail survival and distribution have close relations with micro ecological environment factors,such as vegetation,soil,water and light intensity.
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Objective To investigate the clinical features of gynecological malignant tumor related multiple primary malignant neoplasms (MPMN).Methods Apply retrospective and comprehensive analysis to the clinical data of 30 patients with gynecological malignant tumor related MPMN.Results Synchronous MPMN were found in 9 patients.Their average age was 50.2 years old and their median age was 49 years old.The neoplasms were located at ovary,uterus,cervix,breast and intestine.Metachronous MPMN were found in 21 patients.Their average age was 57.7 and their median age was 57 years old.The median interval between the first and the second primary malignant neoplasm was 4.0 years.The neoplasms were located at breast,ovary,uterus,gastrointestinal tract,uterine cervix,lung etc.In 30 cases,26 of them were treated by surgical operation and further adjunctive treatment of chemotherapy and (or) radiotherapy was conducted as per the neoplasm staging and its pathological results.The rest 4 patients (first primary malignant neoplasms were excised from 3 of them and another one was not treated by surgical operation) received adjunctive treatment of chemotherapy and (or) radiotherapy.Followed ups,which varied from 6 to 60 months,were made to 29 patients and 20 out of the 29 were alive.5-year survival rate of patients with gynecological malignant tumor related MPMN was 47.8％,2-year survival rate was 73.9％,and 1-year survival rate was 88.6％.Conclusion Pay more attention to the patients with gynecological malignant tumor related MPMN,examine the high-risk patients with malignant tumor comprehensively,identify whether it is recurrence,metastasis or new growth of malignant neoplasm,and further ensure early diagnosis and proper treatment,avoiding misdiagnosis and missed diagnosis.
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Objective To study the seasonal changes of glucose levels per unit soft tissue of Oncomelania hupensis. Meth-ods O. hupensis snails were collected from the beach of the Qingyi River in Wuhu City,Anhui Province from August 2012 to July 2013. They were kept in minus 80℃refrigerator immediately. The male snails were distinguished from female,and their soft tissues were collected separately after crushing their shells. The hexokinase method was used to measure the glucose concen-trations,and the results were analyzed statistically. Results The contents of glucose decreased from March to July. The lowest glucose content in the female was 1.87μg/mg in March,and that in male was 3.70μg/mg in July. Both of them increased from August and reached peak levels in September(♀=57.38μg/mg,♂=44.39μg/mg),and then gradually decreased from Octo-ber to next January and increased in February. Conclusion The contents of glucose have seasonal changes regularly in O. hu-pensis.
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Objective To find out a quick,simple and convenient method of determining the viability of Oncomelania hupen-sis. Methods O. hupensis snails were stained for 30 minutes by 0.05%water soluble dye neutral red,0.5%methylene blue,red ink,methylene blue-eosin-borax(MEB)and 0.4%trypan blue,respectively. The soft tissue samples of the snails were observed by a stereoscopic microscope after crushing their shells. Results The vital snails were stained and the dead were unstained in the neutral red. The vital and dead snails were unstained in methylene blue. However,the vital and dead snails were stained in red ink. The partial vital and dead snails were stained in MEB. The vital snails were stained and the partial dead were stained in trypan blue. Conclusion The use of 0.05%water soluble dye neutral red is simple,rapid and accurate in determination of the viability of O. hupensis.
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Objective To investigate the histological distribution of phenol oxidase (PO) in body of Oncomelania hupensis. Methods Oncomelania hupensis snails collected from the riverside of Wuhu section of the Yangtze River were anatomized and the unimpaired software organs were sliced in frozen condition to get the continued sections, then the sections were incubated in pH 6.8 phosphate buffer containing 25 mmol/L catechol at 37 ℃ and investigated systematically for histological distribution of PO in snail bodies. Results The light microscope showed that there were black grannles of PO (histochemical reaction) in the liver, ctenidium, head-foot, tentacle and mantle collar of snail body. Conclusion PO exists in Oncomelania hupensis and locates in the liver, mantle collar, head-foot, tentacle and ctenidium.
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Objective To understand whether phenol oxidase (PO) existed in the body of Oncomelania hupensis and to observe histological distribution of the enzyme. Methods Enzyme histochemical method: adult female and male snails collected from the riverside of Yangtze River of Wuhu section were anatomized and consequently incubated in pH 6.8 phosphate buffer containing 25 mmol/L catechol at 37 ℃. Then under a stereo microscope, some of the snails were observed and recorded for the existing condition and histological distribution of PO in their bodies. Fluorescent histochemical method: after the process of incubation mentioned above, catechol were exsucted from the group of other snails. Afterward, some phosphate buffer solution (pH 6.8) containing 0.05% sodium penobarbital was added into the group. Then the existing condition and histological distribution of PO were also observed and recorded under a fluorescent microscope. Results The first method indicated that the color of the liver surface turned from French grey to light grey in 5 minutes, to grey in 15 minutes and black in 30 minutes. The second method showed that fluorescien appeared on the surface of the snail liver. Conclusion PO exists in the bodies of male and female snails and locates in their livers.