Organoids are three-dimensional (3D) biological structures constructed in vitro by stem cells, which partially mimic the function of real organs. Brain organoids are an extremely important branch of organoid research. This technology can differentiate pluripotent stem cells into the required cell types in a 3D culture environment, and self-assemble into structural bodies, but it is currently unable to fully replicate the structural and physiological features of the real human brain. The maturity of brain organoids may form consciousness, which poses ethical issues such as determining moral status and informed consent in brain organoids research. This paper elaborated on the research progress and future development direction of brain organoids, and proposed multidimensional governance strategies for ethical issues faced in brain organoids research from the perspectives of ethical principles, ensuring public informed consent, and legal supervision. By exploring the above issues, reference will be provided for formulating ethical principles to guide the research and application of brain organoids in the future.

OBJECTIVE: To investigate the gene mutation occurved in AML patients with 29 kinds of fusion genes and 51 kinds of tumor gene. METHODS: Next-generation sequencing (NGS) was used to detected the 49 kinds of targeted gene. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutation were detected by DNA-based PCR and Sanger sequencing. Twenty-nine kinds of fusion genes were dected by multiplex nested RT-PCR. RESULTS: The total gene mutation rate was 91% (109/121) in all the 121 patients. On average, 2.1 mutated genes per patient were identified, among these 121 patients, coexistence of ≥ 3 mutations was frequent (34.7%). The most commonly mutated genes were NRAS (23.96%, n=29), followed by NPM1 (14.04%, n=17), CEBPA double mutations (14.04%, n=17), KRAS (11.57%, n=14)，FLT3-ITD (10.74%, n=13), CSF3R (10.74%, n=13), TET2 (9.92%, n=12) and IDH1 (9.1%, n=11). Overall, fusion genes were detected in 47 (37.3%) patients, including AML/ETO (n=12), CBFβ/MYH11 (n=11), PML/RARa (n=12), MLL rearranagement realated mutation MLL-X (n=10). TLS/ERG (n=1) and DEK/CAN (n=1) in an order of decreasing frequency. Patients with normal karyotype (NK)- AML exhibited more mutations in CEBPA, NPM1, TET2, RUNX1 and IDH1, comparing with abnormal karyotype patients. KRAS mutation in abnormal kayotype patients was significantly higher than that in normal kayotype patients (P=0.014). TP53 mutations were predominantly associated with complex cytogenetics (P=0.199). KRAS mutations were more frequent in core binding factor (CBF) acute myeloid leukemia (AML) and 11q23/MLL rearrangement leukemia, compared with NK-AML (P=0.006 and 0.003, respectively). KIT mutations predominated in CBF-AML (P=0.006). JAK2V617F mutations were detected in two patients and co-occurred with AML-ETO fusions. CONCLUSION At least one mutation is observed in more than 90% patients. On average, more than 2 mutated genes per patient are identified. Some gene mutations are associated with gene rearrangement.
Chromosomal Proteins, Non-Histone ; Genomics ; High-Throughput Nucleotide Sequencing ; Humans ; Leukemia, Myeloid, Acute ; Mutation ; Oncogene Proteins ; Poly-ADP-Ribose Binding Proteins ; Prognosis

OBJECTIVE To investigate the effect of succinate dehydrogenase complex subunit A (sdha)gene on cell proliferation,cell cycle and apoptosis of mouse hepatic cell line BNL CL.2 cells. METHODS The BNL CL.2 cells were transfected by two kinds of sdha-shRNA lentivirus to knockdown sdha gene. The infection efficiency of BNL CL.2 cells infected with lentiviral vectors was analyzed by flow cytometry. The expression of sdha gene and SDHA protein was detected by real-time PCR and Western blotting,respectively. The effect of sdha gene on cell proliferation of BNL CL.2 cells was examined by growth curve,while cell cycle and apoptosis were analyzed by flow cytometry. RESULTS The infection efficiency of BNL CL.2 cells in sh-control group and in sdha-shRNA group was above 80%. Compared with sh-control group,the expression of sdha gene in BNL CL.2 cells infected with sdha-shRNA lentivirus was decreased by about 20 times(P<0.01),the expression of SDHA protein was decreased by about 10 times(P<0.01),and the growth rate was about 70%that of sh-control group(P<0.05). The cells were arrested in S phase,and the percentage of cells in S phase was 0.74 times that of sh-control group(P<0.01). The percentage of cells in G0/GI phase was 1.17 times that of sh-control group(P<0.01). The percentage of cells in G2/M was 1.37 times that of sh-control group(P<0.01). But there was no obvious difference in the apoptosis rate. CONCLUSION The reduced expression of SDHA protein can inhibit the proliferation of mouse hepatic cells,and the inhibitory mechanism may be cell cycle arrest. There is possibly no relationship between inhibition and cell apoptosis.

BACKGROUND: Both density gradient centrifugation and adherence method arc frequently used to isolate bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the approaches to isolate, culture and identify the rabbit BMSCs in vitro by the combination of den,ity gradient centrifugation and adherence method. DESIGN, TIME AND SETTING: Contrast cytological study, which was performed in Central Laboratory of Shanghai 6th People's Hospital between October 2007 and March 2008.MATERIALS: Six 2-week-old rabbits were selected for BMSCs preparation and primary culture; Percoll separating medium (1.073 kg/L) was also used for this study.METHODS: BMSCs were separated and purified with Percoll separating medium by density gradient centrifugation and adherence method. The three-, five-, seven-, and nine-passage BMSCs were counted for growth curve. MAIN OUTCOME MEASURES: Morphological features and growth states of primary and passage cells were observed under inverted microscope. Indirect immunofluorescence of CD44 and CD34 antibodies were used to examine the stem cells. CD44 staining was positive, and CD34 staining was negative, suggesting the extracting and purifying cells were BMSCs. RESULTS: The passage BMSCs were uniformly distributed like fusiform shape, which were more uniform than primary cultured cells. The BMSCs grew productively and proliferated rapidly; meanwhile, the nucleolus was clear, caryopla.sm was in a large proportion, morphological features were uniform, ceils like bostrychoid or whirlpool were arranged parallelly, and the five-pa.ssage cells were not changed remarkably. Proliferation was decreased gradually with the passage increasing; especially, the proliferation of three-five-passage cells was the strongest. The separated cells expressed CD44 but not CD34. CONCLUSION: High-purified rabbit BMSCs are obtained by both density gradient centrifugation and adherence method.