Objective To establish a culture method for micropapillary lung adenocarcinoma organoids and conduct targeted drug screening.Methods Organoids were extracted and cultured from a surgical tissue sample of a patient diagnosed with micropapillary lung adenocarcinoma,and the growth of lung cancer organoids was observed and recorded dynamically.The morphological and gene expression characteristics of tumor cells between lung cancer organoids and parental tissue were compared using hematoxylin eosin(HE)staining and immunohistochemical methods.Real time fluorescence quantitative polynucleotide chain reaction(qRT-PCR)method was used to detect gene mutations in lung cancer parental tissue and organoids.Finally,based on results of genetic testing,targeted drugs were selected and their therapeutic effects were verified.Results We have successfully cultured spherical organoids from micropapillary lung adenocarcinoma tissue,which can be passaged for at least 3 generations.HE staining results showed that the morphology of tumor cells in organoids was roughly consistent with that of parental tissue.The immunohistochemical results showed that the protein expression levels of various genes in lung cancer organoids and parental tissue were roughly the same.Results of gene mutation analysis showed that the mutated genes in lung cancer parental tissue and organoids were consistent,both reflecting RET fusion.The screening results of targeted drugs based on lung cancer organoids showed that vandertinib had the best anti-tumor effect in vitro.Conclusion Drug screening experiments based on micropapillary lung adenocarcinoma organoids can screen highly efficient targeted drugs in a short period of time,which may benefit patients with micropapillary lung adenocarcinoma.

Objective:To summarize the clinical manifestations, diagnosis and surgical treatment of abdominal unicentric Castleman's disease.Methods:The clinical data of abdominal unicentric Castleman's disease cases admitted to the General Surgery Department of the First Affiliated Hospital,Nanjing Medical University from Jan 2009 to Dec 2019 was retrospectively analyzed.Results:A total of 18 patients were included with definite pathological diagnosis. The main complaint was abdominal pain and discomfort (50%, 6/12), dizziness and fatigue (25%, 3/12), multiple rash with oral ulcer (16.7%, 2/12) and weight loss (8.3%, 1/12). All patients received surgical resection. Postoperative pathology showed clear vascular type in 15 patients and plasma cell type in 3 patients. There were no major complications nor mortality.Conclusion:Abdominal unicentric Castleman's disease has diverse clinical manifestations, which was difficult for preoperative diagnosis, postoperative prognosis is satisfactory.

Objective:To observe the ferroptosis in hippocampal neurons of rats with multiple cerebral concussion (MCC) .Methods:Ninety clean grade male Wistar rats with a body mass of (250±10) g were randomly divided into control group (12 rats) and model group (78 rats) according to the random number table method. The rat of MCC model was prepared by hitting the frontotemporal lobe of rats with free fall method once a day for 3 consecutive days.Then the MCC model rats were randomly divided into 12 h, 24 h, 48 h, 72 h, 7 d and 14 d groups with 12 rats in each group. The balance beam experiment was used to detect the motor coordination function of the rats. The levels of interleukin-β(IL-1β), interleukin-6(IL-6), serum glutathione (GSH), neuron-specific enolase (NSE) in serum of rats were detected by ELISA.The content of iron ion in hippocampus was detected by colorimetry. The mRNA and protein levels of glutathione peroxidase 4(GPX4), ferritin heavy (FTH) and ferritin light(FTL) in the hippocampus were detected by RT-PCR and Western blot. Prussian blue staining was used to observe the iron deposition in brain tissue.The ultrastructural changes of hippocampal neurons and mitochondria were observed by transmission electron microscope. SPSS 24.0 statistical software was used for one-way ANOVA among groups, and LSD test was used for multiple pairwise comparison.Results:In the balance beam experiment, the passing time and motor coordination score of rats in each group were significantly different ( F=30.08, 60.34, both P<0.05). The passing time and motor coordination score of rats in the 48 h group ((87.00±4.74) s, (4.75±0.43)) were significantly higher than those in the control group ((35.13±6.99) s, (0.75±0.23)) (both P<0.05). There was significant difference in the total iron ion content, Fe 2+ content and Fe 3+ content in hippocampus of rats in each group ( F=25.20, 94.42, 40.25, all P<0.05), and the content of Fe 2+ in hippocampus of 48 h group was significantly higher than that of the control group ((10.17±0.05) ng/μL, (8.65±0.01) ng/μL)( P<0.05). In the results of RT-PCR and Western blot, the mRNA and protein levels of GPX4, FTH and FTL in hippocampus of each group were significantly different ( F=37.94, 82.09, 49.01, 71.63, 28.94, 15.78, all P<0.05). The mRNA level and protein level of GPX4 ((1.09±0.01), (0.23±0.01) )and FTL ((1.60±0.03), (0.64±0.02)) in 24 h group were significantly higher than those of the control group (GPX4: (1.00±0.02), (0.17±0.01)), FTL: ((1.00±0.04), (0.32±0.01))(all P<0.05). The mRNA level and protein level of FTH ((0.24±0.03), (0.07±0.01)) in 24 h group were significantly lower than those of the control group((1.00±0.01), (0.67±0.03))(both P<0.05). The results of electron microscope showed that the hippocampal neuronal cells of the model rats were reduced, the nucleolus was broken and the nuclear membrane was shrunk in varying degrees, the mitochondria were swollen and deformed and there were vacuoles, and the cristae in the mitochondria decreased or disappeared. Conclusion:The levels of inflammatory reaction and oxidative stress in the multiple cerebral concussion model rats increase, and the hippocampal neurons show the characteristics of ferroptosis, especially at 24 h and 48 h.

【Objective】 To analyze the clinical and pathological characteristics of mucinous tubular and spindle cell carcinoma （MTSCC） in the kidney so as to evaluate the value of radiotherapy in local treatment of refractory MTSCC. 【Methods】 A case of MTSCC with recurrent recurrence and metastasis was reported， and the existing literature on MTSCC was reviewed and analyzed to explore the value of radiotherapy in the treatment of MTSCC. 【Results】 After three operations， postoperative pathology of the patient showed mucinous tubular and spindle cell carcinoma of the kidney. Radiotherapy was performed in the first recurrence area， and long-term follow-up in the tumor bed area showed no recurrence. 【Conclusion】 MTSCC， a rare renal epithelial tumor， has the low-grade malignance in most cases， and rarely recurs and has metastasis. This patient had repeated recurrence and metastasis before surgery， but after tumor bed radiotherapy after the first recurrence no lesion was found， suggesting that radiotherapy plays a certain role in preventing local recurrence. It is necessary to further explore the value of radiotherapy in the treatment of MTSCC.

Objective The aim of this study was to investigate the anti-tumor effect on sequential injection of heterogeneic lymphocyte(HL)and autogeneic lymphocyte(AL). Methods The HL was prepared by using CC3HF1 mice as feeders. CB6F1 mice were used as recipients,and Hepa1-6 cells were inoculated into the recepients′groin subcutis. A cryoprecipitate was extracted from mouse plasma by freeze-thaw method to prepare fibrin Glue(FG);FG was combined with HL or AL to be FG-HL or FG-AL. The experimental treatment consisted of two stages. At first stage(15 d),FG-HL were injected on the surface of the tumor-bearing tissue of the recipients as the experimental group,and FG-phosphate buffer saline(FG-PBS)were injected on the surface of the tumor-bearing tissue of the rest recipients as the control group. The immunological factors such as tumor cell killing rate of the spleen lym-phocytes and numbers of lymphocytes,CD8 +T and NK in the two groups were detected,respectively. At later stage(10 d),a part of mice were randomly selected from the experimental and control groups,and the lymphocytes( AL) were used to form FG-AL,which were injected on the surface of tumor-bearing tissues in the rest of mice. Tumors in mice of the two groups were compared for tumor volume and tumor inhibition rate. Results The tumor cell killing rate of AL in the experimental group(26. 70 ± 7. 22) was signifi-cantly higher than that in the control group(5. 70 ± 2. 68)(P<0. 01). Numbers of mouse spleen lymphocytes,CD8 +T cells and NK cells were significantly higher than the corresponding values of the control group(P<0. 05). After the two-stage treatment,the aver-age tumor volume of the experimental group[(1.20 ±0.33)cm3]was significantly smaller than that of control group[(2.05 ±0.37) cm3](P<0. 01). The tumor inhibition rate in the experiment group was 41. 5% when compared to the control group. Conclusion Local injections of FG-HL followed by FG-AL can significantly inhibit the growth of transplanted tumor in mice;it is expected to become an anti-tumor biological therapy.

Objective: To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells. Methods: The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining. Results: Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) μm and 89.44±8.36, (56.56±5.37) μm and 93.84±8.79, as well as (34.57±6.61) μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05). Conclusions Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.

OBJECTIVETo inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).

METHODSWestern blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T. The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry. The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot. The cell growth curve was drawn by MTT test. The Ki-67-positive rate was calculated using immunofluorescence staining. The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.

RESULTSWestern blot results showed that BTG2 relative expression levels were 0.83 ± 0.12, 0.18 ± 0.04, 0.20 ± 0.05 and 0.36 ± 0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively. The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5% (33/40), 77.5%(31/40) and 17.5% (7/40), respectively, with a significant difference between two groups (P < 0.05). Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2 ± 8.0)%, (81.4 ± 9.7)% and (50.1 ± 7.1)%, respectively, showing a significant difference between two groups (P < 0.05). The scratch test results showed that in the control group, empty vector group and BTG2 transfection group, the distance of SW620 cells between two sides was (79.27 ± 11.24) µm, (80.65 ± 12.17) µm and (124.77 ± 19.63) µm, respectively, with a significant difference between two groups (P < 0.05). Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5 ± 13.1)%, (73.2 ± 12.9)% and (47.4 ± 9.1)%, respectively, showing a significant difference between two groups (P < 0.05). The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.

CONCLUSIONSBTG2 gene is involved in colon cancer cell proliferation and metastasis, and effectively restores the function of BTG2 protein. Therefore, it may be expected to become a new option in gene therapy for colon cancer.

B-Lymphocytes ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; genetics ; Colonic Neoplasms ; Genetic Vectors ; Humans ; Immediate-Early Proteins ; genetics ; Immunohistochemistry ; Plasmids ; Transfection ; Tumor Suppressor Proteins ; genetics

Objective To explore the regulation mechanism for miR - 125a - 5P in epidermal growth factor receptor(EGFR)signaling pathway in medulloblastoma. Methods The potential targets of miR - 125a - 5P in the EGFR signaling pathway were predicted by TargetScan and Sanger software,there were 3 groups:control group,non -sense group and miR - 125a - 5P group. Their relationship,between miR - 125a - 5P and cyclin - dependent kinase in-hibitor 2B( CDKN2B),E2F transcription factor 3( E2F3),mitogen - activated protein kinase 14( MAPK14)and growth factor receptor - bound protein 10(GRB10),were tested by luciferase experiments. After miR - 125a - 5P oligo-nucleotide was transfected to D341 cells,miR - 125a - 5P level was detected by reverse transcription polymerase chain reaction. Then the thiazolyl blue tetrazolium bromide assay was used to draw the cell growth curves,and Transwell assay was used to detect cell migration ability. The expression levels of GRB10,EGFR,phosphatidylinositol 3 - kinase(PI3K) and Ras were tested by Western blot method. Results The results of luciferase experimental results showed that GRB10 was the only target gene of miR - 125a - 5P. After miR - 125a - 5P being transfected,the D341 cell prolifera-tion obviously declined markedly. Compared with control group[(38. 16 ± 7. 47)% ]and the non - sense group [(36. 79 ± 8. 94)% ],cell migration rate in the miR - 125a - 5P group was lowest[(13. 59 ± 4. 41)% ],and there was a significant difference among 3 groups(χ2 = 11. 495,P < 0. 05);in the miR - 125a - 5P group,the expression level of EGFR increased 1. 67 times,GRB10,PI3K and Ras levels were reduced to 23% ,61% and 42% . Conclusion miR - 125a - 5P can inhibit tumor growth by silenced GRB10 expression targeting EGFR downstream signaling pathways in medulloblastoma.

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.

Objective To inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).Methods Western blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T.The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry.The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot.The cell growth curve was drawn by MTT test.The Ki-67-positive rate was calculated using immunofluorescence staining.The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.Results Western blot results showed that BTG2 relative expression levels were 0.83±0.12,0.18±0.04, 0.20±0.05 and 0.36±0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively.The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5%(33/40), 77.5%(31/40) and 17.5%(7/40), respectively, with a significant difference between two groups (P<0.05).Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2± 8.0)%, (81.4±9.7)%and (50.1±7.1)%, respectively, showing a significant difference between two groups ( P<0.05 ) .The scratch test results showed that in the control group, empty vector group and BTG2 transfection group , the distance of SW620 cells between two sides was ( 79.27 ±11.24) μm, ( 80.65 ± 12.17) μm and (124.77±19.63) μm, respectively, with a significant difference between two groups ( P<0.05) .Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5±13.1)%, (73.2±12.9)%and (47.4±9.1)%,respectively, showing a significant difference between two groups ( P<0.05) .The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.Conclusion s BTG2 gene is involved in colon cancer cell proliferation and metastasis , and effectively restores the function of BTG2 protein.Therefore, it may be expected to become a new option in gene therapy for colon cancer.