| Hemifacial microsomia, following cleft lip and palate, is a common clinicalmaxillofacial malformation disease. Deformity mainly related to maxillofacial region, thedisease seriously affect the patient’s daily life and mental health.The diagnosis and treatment of the disease have been focused by imaging,orthodontics and maxillofacial surgery. However, due to the complex clinicalmanifestations and unknown etiology, the diagnosis in addition to the collection ofdetailed family history and personal medical history is depend on detailed clinicalexamination. In recent years, with the rapid development of Stomatology, orthodontics andorthognathic surgery, the treatment of HFM patients has been promoted.The cause of the disease has been also a hot topic.There is no conclusion, but most ofthe researchers believe that HFM is possiblely caused by a variety of causes, including genetic defects, teratogenic, and vascular malformations. Most studies confirmed that thedisease is a hereditary disease due to genetic mutations. And the determination of thecausative gene has become an urgent problem. Researchers have proposed a variety ofgenes, but were overthrew by the later study. Some scholars have recently proposedTCOF1as disease gene, but whether explicitly need further confirmed.As we all know, BMMSCs play an important role in the process of growth anddevelopment. The current study has been proved, BMMSCs not only in bone formation,but also plays an important role in immune regulation. Increasing number of studies haveconfirmed that a defect in the gene as well as the microenvironment changes can influencethe biological behavior, differentiation and function of stem cells, resulting indevelopmental defects. Now studying on stem cells from patients has been veryimportant on genetic diseases research.Objectives1. To collecte patients with Hemifacial microsomia.2. To verify TCOF1gene for the accuracy of the genes that cause the disease.3. To explore the difference of the biological behavior and differentiation capacitybetween hBMMSCs from the affected jaw and healthy side.4. To explore the difference of immune regulatory capacity between hBMMSCs fromaffected maxillary and unaffected side.Methods1. We unified diagnostic criteria after comprehensive and exhaustive inquiry andclinical examination, resulting in a collection of14HFM patients using OMENSclassification based on the clinical features and organ involvement. In the case of patientsand their families informed consent, we extracted the patients2ml vein, extracted thegenome-wide DNA, and verified the reported suspicious pathogenic genes TCOF1bysequencing its26exons.2. In the case of patients and their families informed, we harvested the tissue ofaffected side and the unaffected side of mandibular, tissue blocks were isolated andcultured hBMMSCs. And with normal mandible hBMMSCs as normal control group, the difference of the cells in each group was studied. Flow cytometry expression of surfacemolecules; clone colony formation, MTT, EDU detection, cell cycle, apoptosis analysiswere performed to detect cell proliferation ability. Osteogenic, adipogenic, and neuralinduction detection hBMMSCs multipotent differentiation capacity. Real time-PCR andWestern Blot analysis were performed to detect the osteogenic and adipogenic iconicgenes expression. As well as the different groups hBMMSCs TA/TCP mixed implantedsubcutaneously into nude mice, harvested after8weeks, fixed decalcified the two weeksparaffin-embedded sections, HE staining observation to detect in vivo osteoid formation.3.Using an ELISA to detect the above groups’ hBMMSCs culture supernatant ofINF-γ, IL-6, IL-10and IL-17expression, Real time RT-PCR was performed to explore theexpression changes of these validation factors in each group. We co-cultured hBMMSCsdirectly with T lymphocytes, examined the T lymphocytes cycle after co-culture, andobserved the proliferation of cells in each group of T lymphocytes. The DSS ulcerativecolitis mouse model was built, and hBMMSCs were injected once on the third day afterthe model building. Mice were observed daily about the body weight, stool, disease status.Mice were killed on the tenth day, and the colonic pathology was observed in order todetermine the outcome of hBMMSC treatment.Results1. Due to the complexity of the HFM disease and its similarity with the phenotype ofmultiple other Maxillofacial genetic disease, we collected14patients using the OMENSclassification after detailed examination. We found that even in the same patients, theextent of involvement of various organs was different. Measured by exon sequencing, wecompared the nucleic acid sequence homology and exclude the SNP loci. In conclusion,we do not think TCOF1is the pathogenic gene of HFM.2. Each group of the mandible hBMMSCs expressed the mesenchymal surfacemarkers, without expressing hematopoietic markers. Clonal colony, MTT, EDU detection,flow cytometry results show the proliferation capacity of hBMMSCs from affected sidewas lower than that of normal controls. Osteogenic, adipogenic, and induced todifferentiate into cartilage had proved that cultured hBMMSCs hold the ability to multi-differentiation. Comparison of the cells in each group and found no significantdifferences between groups of cells’ neural differentiation. However, after osteogenic andadipogenic induction, the differentiation capacity of hBMMSCs from affected side wasweaker than the normal controls from the RNA and protein levels. The differentiationcapacity between hBMMSCs frome normal side of HFM patients and normal people werefairly consistent.3. Using ELISA to detect the culture supernatant of each group hBMMSCs, the levelof expression of inflammatory cytokines of hBMMSCs frome affected side wassignificantly higher. Real time RT-PCR detecting RNA levels of hBMMSCs also obtainedsimilar results. We detected the T-cells cycle co-cultured with hBMMSCs. The resultsshowed that the inhibitory effect on T cell proliferation caused by hBMMSCs fromaffected side was less than the normal control groups. Treating ulcerative colitis modelmice by different types of hBMMSCs, we observed that the immune suppression ability ofhBMMSCs from affected side was declined, while the immune suppression ability ofhBMMSCs frome normal side of HFM patients was similar with that of hBMMSCs fromenormal people.ConclusionThrough case studies, we found that even same patient with HFM, its degree ofinvolvement of the various organs was not consistent. TCOF1gene reported by studieswere not the virulence gene of HFM, and the virulence gene must be explored further.The proliferation of hBMMSCs from patients’ health side is similar to normal controlgroup, while the proliferation of hBMMSCs from patients’ affected side was relativelylower. The osteogenic and adipogenic differentiation capacity of hBMMSCs from affectedside was weaker than the normal controls. The osteogenic and adipogenic differentiationcapacity between hBMMSCs frome normal side of HFM patients and normal people werefairly consistent. No significant differences between groups of cells’ neural differentiationin each group was found. The immune suppression ability of hBMMSCs from affectedside was declined, while the immune suppression ability of hBMMSCs frome normal sideof HFM patients was similar with that of hBMMSCs frome normal people. |
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