بحث لإيجاد خلايا بيتا المصنعة للإنسولين من بطانة الأمعاء كعلاج جذري يعوض عن زرق الإنسولين لمرضى السكري النمط الأول

تثبيط عامل النسخ المعروف فوكسو 1 (FOXO1) يولد خلايا تنتج الإنسولين في نموذج زرع أسلاف خلايا بطانة أدمة الأمعاء للبشر. 

30 June 2014

الخلاصة

يبقى إستحداث مصادر بديلة عن خلايا بيتا المنتجة للأنسولين هدف معالجة مرضى السكري. في حين أنَّ معظم الجهود توجَّهت إلى إنتاج الخلايا الجنينية أو الخلايا الجذعية المحفزة الى خلايا مماثلة لخلايا بيتا المنتجة للإنسولين أظهرت البحوث أنَّ أسلاف خلايا بطانة أدمة الأمعاء التي تتمتع بميزات الغدد الصماء عند الفئران يمكن أن تحويرها الى خلايا منتجة للانسولين بالإستجابة الى تحفيز سكر الدم ومن خلال إجتثاث عامل النسخ المعروف فوكسو 1 (FOXO1). علماً بأنَّ هذا العامل موجود في أسلاف خلايا بطانة أدمة الأمعاء وفي الخلايا المنتجة للسيروتونين. وباستخدام طرق مختبرية يمكن تكاثر خلايا بشرية منتجة للإنسولين ومُعبَّر جينياً فيها عن علامات خلايا بيتا الناضجة لغدة البنكرياس وتستطيع إفراز الإنسولين وكذلك الببتايد س الأمر الذي يُبقي هذه الخلايا نشطة في جسم الفئران بعد أنْ تُزرع فيها. والبحوث الجارية تهدف الى تطبيق هذه الطرق لزرع خلايا منتجة للإنسولين كعلاج لمرض السكري عند البشر.

Abstract

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors, we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive, insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells, we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded small hairpin RNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells, release C-peptide in response to secretagogues and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes.

مشروع نظام تحرير إنْكابترا (فياسايت “ViaCyte”) الذي يهدف الى إختبار وسيلة لمعالجة جذرية لمرضى السكري النمط الأول بالخلايا الجذعية .

نبذة عن البحث: ضمن علم الطب التجديدي  (Regenerative medicine) يعتمد العلاج على تحفيز الخلايا الجذعية الجنينية (Embryonic stem cells) في إنبوبة المختبر لتتحول إلى خلايا منتجة للإنسولين ثم توضع داخل كبسولة صغيرة  يتم زرعها تحت الجلد ويُعرف هذا النظام بنظام تحرير إنْكابترا   (Encaptra delivery system) . الكبسولة تحمي الخلايا من التلف الذي يسببه جهاز مناعة المريض ضد هذه الخلايا والذي يشكل الحاجز الذي يُعيق المشاريع البحثية في هذا المجال. وفي هذه التجارب أمكن وبعد 12 أسبوعا من زرع الكبسولة بشكل مناسب قرب الأوعية الدموية بقيت الخلايا ما زالت الخلايا تنتج الأنسولين وكذلك يتضاعف عددها ولم تظهر أية آثار جانبية.  يعمل الباحثون الأكاديميون والشركات على إيجاد وسيلة لشفاء مرضى السكري النمط الأول بإتباع مختلف الطرق وتعتبر هذه الطريقة الأولى من نوعها التي تمَّ تجربتها على المرضى. وإذا نجحت هذه الطريقة فسوف تكون متوفرة لمرضى السكري النمط الأول خلال عدة سنوات قادمة وتمهد الطريق أيضاً لمعالجة مرضى السكري النمط الثاني.

Cell rearrangement in transplanted human islets 2016

Published online before printNovember 3, 2015, doi:10.1096/fj.15-273805February 2016The FASEB Journalvol. 30 no. 2 748-760

Abstract

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central β cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and β cells into islet-like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti-VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.—

Lavallard, V., Armanet, M., Parnaud, G., Meyer, J., Barbieux, C., Montanari, E., Meier, R., Morel, P., Berney, T., Bosco, D. Cell rearrangement in transplanted human islets.

The 7 important molecular tools used in genetic engineering

Gene cloning is the process in which a gene of interest is located and copied  out of DNA extracted from an organism. When DNA is extracted from an organism, all of its genes are extracted at one time. This DNA, which contains thousands of different genes. An engineer is a person who constructs and manipulates according to a plan. The term genetic engineer is for an individual who is involved in genetic manipulations.They must find the one specific gene that encodes the specific protein.

The genetic engineer’s molecular tools namely the enzymes most commonly used in genetic engineering experiments are given below:

1. Polymerase Chain Reaction

The discovery of thermostable DNA polymerases, such as Taq Polymerase, made it possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. Primers specific to a particular region of DNA, on either side of the gene of interest, are used, and replication is stopped and started repetitively, generating millions of copies of that gene. These copies can then be separated and purified using gel electrophoresis.

2. Restriction Enzymes (Molecular Scissor)

The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments, of varying sizes. These can be separated using gel electrophoresis or chromatography.

3. Electrophoresis

Purifying DNA from a cell culture, or cutting it using restriction enzymes wouldn’t be of much use if we couldn’t visualize the DNA – that is, find a way to view whether or not your extract contains anything, or what size fragments you’ve cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts.

4. DNA Ligase

In genetic research it is often necessary to link two or more individual strands of DNA, to create a recombinant strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5′ ends, respectively.

5. Selection of Small Self-Replicating DNA

Small circular pieces of DNA that are not part of a bacterial genome, but are capable of self-replication, are known as plasmids. Plasmids are often used asvectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together generating what is known as a recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, recombinant plasmids containing bacteriophage genes.

6. MethodtoMove a Vector into a Host Cell

The process of transferring genetic material on a vector such as a plasmid, into new host cells, is called transformation. This technique requires that the host cells are exposed to an environmental change which makes them “competent” or temporarily permeable to the vector. Electroporation is one such technique. The larger the plasmid, the lower the efficiency with which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus or other viral vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but may cause insertion of DNA in parts of our chromosomes where we don’t want it, causing complications and even cancer.

7. Methods to Select Transgenic Organisms

Not all cells will take up DNA during transformation. It is essential that there be a method of detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance and transgenic cells can be selected based on expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively.

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ماهي الأسباب التي تساهم في زرع المخاوف غير المُبررة علمياً من إستعمال الإنسولين (PDF)

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