| Background and ObjectivesLipoma is one of the most common benign surface tumors in the clinic.It can occur in any part of the body,consisting of single or multiple flat masses of varying sizes.The lipoma is round,elliptical or irregular,and can be divided into a multi-lobed shape by the interval formed by the fibrous tissue.The tumor volume is generally small and the surface skin is normal.When the tumor is large,the local swelling,ups and downs.The clinical characteristics are slow onset and progress,mild symptoms and local soreness occurs during physical activity occasionally.At present,most of the treatment methods of lipoma are surgical resection,and the curative effect is good after resection,generally no recurrence.Therefore,the disease has not attracted the attention of academia for a long time.But,with the increase of living pressure,the dietary structure gradually inclining to high protein and high fat,and the increase of unhealthy living habits brought by social progress,the incidence of lipoma is increasing year by year.If the tumor grows in the exposed area,the scar left by the surgery may affect the patient’s daily life and social interaction.Multiple lipomas of the whole body cannot be totally removed by surgery,and patients will be worried about their continuous progress but unable to find a radical cure,resulting in other psychological disorders.There are also some other special types（such as intraspinal,intracranial,and renal）lipomas.If not treated in time,the tumor gradually enlarges and oppresses important blood vessels and nerves,which will cause irreversible organ dysfunction and even endanger the patient’s life.If surgical treatment is performed,the anatomical structure around the mass is complicated,the operation is extremely difficult,and postoperative complications are more.Based on the above background,some scholars have turned their attention to other therapeutic areas beyond the open surgery of lipoma,including interventional surgery,tumor injection therapy,traditional Chinese medicine treatment,etc.,but the effect is not ideal.There is insufficient basic research on related pre-orders,it seems to be no new breakthrough in the treatment of lipoma.According to literature review,as early as 1958,Hewitt[1]et al.found that after transplanting tumor cells of leukemia mice into the same kind of mice,only 0.1%-1%of the few cells formed clone colonies.The researchers proposed a theory to explain the finding:tumor cells are functionally heterogeneous,only a limited number of tumor cells have the ability to produce tumors,and there was a high probability that these tumor cells would continue to proliferate and differentiate into the same or similar tumor cells.Therefore,these cells（tumor stem cells）with high proliferation,differentiation and tumorigenicity had become a research hotspot.In 2007,Lin et al.based on the theory of cancer stem cells,first isolated lipoma stem cells（LSCs）and described their morphology,and mentioned that LSCs are homologous to normal adipose-derived stem cells（ADSCs）and have some correlation[2].ADSCs are adult pluripotent stem cells,which are present in human normal adipose tissue and have strong proliferation and differentiation ability.They are one of the emerging seed cells of tissue engineering,its related basic research is still in infancy,it is mentioned in the literature that ADSCs are closely related to soft tissue tumors such as lipoma and liposarcoma.Based on the theory of cancer stem cell theory,this study attempts to find out the differences in morphology,biodynamics,and bioinformatics between LSCs and ADSCs,so as to understand the changes in lipoma relative to normal adipose tissue.The purpose is to 1.improve the experimental methods and principles of isolation and culture of adipose-derived stem cells.2.complete the basic experimental research related to lipoma.3.find the key bioinformatics nodes for the formation of LSCs.Methods:1.Taking ADSCs as an example,the enzyme digestion was used to isolate and culture ADSCs,to verify their stemness,to confirm the effectiveness of enzymatic digestion to obtain adipose tissue derived stem cells and to compare the proliferation and differentiation ability of ADSCs from different age groups.According to the results,suitable lipoma and fat specimens were screened:Five different age groups（12,21,31,41,68 years old）were selected for adipose tissue.According to the standard procedure of extracting stem cells by enzymatic digestion,the target cells were isolated and subcultured,the morphological characteristics and differences of P2 were observed.Immunofluorescence staining was used to compare the proportion of ki67 stained nuclei of 5 groups of cells at P3.Alizarin red staining and oil red O staining were used to verify their multidirectional differentiation ability and to confirm the stemness of target cells.Five groups of cells transferred of culture to P5 were stained with B-gal to observe the effect of age on the activity of ADSCs.2.The LSCs were isolated and compared with ADSCs in morphology,and the two cells were initially compared to analyze the difference from the perspective of growth kinetics such as proliferation and differentiation speed and aging rate:Referring to the part I of the experimental results,suitable lipoma and adipose tissue blocks（excluding age,BMI,basic diseases and other interfering factors）were selected to obtain LSCs and ADSCs by enzymatic digestion.The adipogenic,osteogenic and chondrogenic differentiation of ADSCs and the flow cytometry which used to analyze surface marker expression were tested to confirm that the obtained cells were the target cells.They were inoculated and subcultured to observe the morphological differences between the two at different passage stages.The BCIP/NBT staining was used to compare the stemness,the immunofluorescence experiment was used to compare the proliferative activity,the plate counting method was used to compare the proliferation rate（difference of stemness was verified on the side）.3.Comparing LSCs and ADSCs by bioinformatics methods,to understand the cellular components that play an important role in the development of lipoma,as well as related signaling pathways,biological processes,etc.:The LSCs and ADSCs were subjected to RNA extraction and reverse transcription.RNA-seq was performed on two types of samples using a transcriptome sequencing technology based on Illumina high-throughput sequencing platform,Both gene expression were analyzed.Then GO analysis and KEGG signaling pathway analysis were carried out on differentially expressed genes,to understand the biological processes,cellular components,molecular functions,signaling pathways,etc.of LSCs compared with ADSCs.Results:1.Under the inverted microscope,the cells obtained by enzymatic digestion were full spindle-shaped,and with the increase of age,the cells gradually appeared flat and adherent,suggesting aging changes.Immunofluorescence staining of the obtained cells showed that Ki67 red staining had a tendency to proliferate,also tended to decrease with the age of the specimens.The above cells were cultured in osteogenic medium for 14days and after stained with alizarin red.It was observed that calcium salt deposition（calcified nodules）was typical,suggesting that the above cells had osteogenic differentiation.The cells were cultured in adipogenic medium for 14 days,and after oil red O staining,a large amount of red stained particles（lipid droplets）appeared in the cells under the microscope,and it was confirmed that the cells had undergone adipogenic differentiation.Multi-directional differentiation experiments confirmed that the cells extracted by enzymatic digestion were ADSCs,and the differentiation ability of ADSCs tends to weaken with age.B-gal staining also confirmed that as the age increases,the number of senescent cells in the stem cells gradually increases.2.LSCs and ADSCs were successfully isolated.In the three differentiation experiments of ADSCs,ADSCs were identified by toluidine blue staining and chondrogenic differentiation was observed after 3 weeks of culture.The results of flow cytometry showed that the expressions of surface markers such as CD44,CD73,CD90and CD105 were consistent with the literature.In the morphological comparison between the two groups,the newly isolated SVF cells were more numerous and denser in the ADSCs group,while the number of LSCs was smaller.In cell adherence and subsequent passage,LSCs group had different cell morphology and size,while ADSCs group showed more orderly cell arrangement in morphology,and there were obvious differences between the two groups.The results of BCIP/NBT staining indicated that the osteogenic differentiation of LSCs was faster than that of ADSCs,but the morphology of differentiated cells showed significant differences with ADSCs,suggesting that the differentiation may be faster than normal osteogenesis.Two stem cell groups were placed in the plate culture,and more colony formation was observed in the LSCs after 48 hours.3.The RNA-seq project speculated that the up-regulated genes in differentially expressed LSCs were mainly related to the growth and differentiation of tumors.GO analysis showed that in the LSCs group,the genes of molecular functions,cell components and biological processes related to cell growth and proliferation,intracellular activity were significantly up-regulated,and genes related to the secretion of tumor-promoting factors and anti-tumor effects of cells also appeared.KEGG signaling pathway analysis also indicated abnormal up-regulated genes of related signaling pathways such as mucin-type O-glycan,and indicated that LSCs tend to invade surrounding tissues.ConclusionThe target cells obtained by enzymatic digestion are indeed ADSCs needed for research.They can have stable transfer in culture with good multidirectional differentiation ability.Such stem cells have a tendency to age with aging process.The preliminary morphology and growth kinetics of the LSCs and ADSCs were compared.It can be clearly seen that the LSCs grow faster than the ADSCs,the proliferation rate is fast,and the differentiation trend is strong,but they have obvious atypia,and the proliferation,passage and differentiation of corresponding cells also show different degrees of heterogeneity.The results of bioinformatics comparison suggest that compared with ADSCs,LSCs have more active intracellular activities,stronger growth and proliferation levels,and similar metastasis,invasion,abnormal differentiation,and immune escape of some malignant tumors.The biological processes,cellular components,molecular functions,signaling pathways involved in it are worthy of our in-depth discussion in future researches. |
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