IDENTIFICATION, ISOLATION, PURIFICATION, AND CARACTHERISATION OF

PANCREATIC CELLS IN TYPE I DIABETIC PATIENTS

by John Nelson

BACKGROUND

One in every 14 Americans has diabetes, and another 40% of the population are at risk
for developing the disease. Every year, diabetes accounts for more than 200,000 deaths,
82,000 amputations, and 44,400 new cases of end-stage renal disease and up to 24,000
new cases of blindness in the United States (1). Then diabetes is one of most severe
diseases in human beings in spite of type II diabetes is undiagnosed in nearly 50% of
persons with the disease. After cardiac disease, cancer, and blood pressure, diabetes
remains a high prevalence disease whose medical findings are so far minimal. Diabetes
may be caused by viral infections, pancreatic cell intoxication or genetic forms.

The purpose of this study design is to allow for a best scientific knowledge of action
mechanism against diabetes and for a relief by the means of research the warning
population.

STUDY DESIGN

a) Fresh pancreatic cells from human organs (Transplantation Unit)

b) Langerhans islets (identification from glucagon pancreatic cells)

c) Isolation of insulin pancreatic cells by chromatography: HPLC (High pressure liquid

chromatography) to measure insulin production (diabetes?); and affinity chromatography
to purify

d) Purification of pancreatic cells by affinity chromatography

e) Storage of insulin pancreatic cells

f) Re-activation of pancreatic cells in culture medium; Sensitization via activator drugs

g) Identification of DNA from pancreatic cells

h) Isolation of DNA

i) Utilization of molecular techniques (2):

PCR (Polymerase Chain Reaction) and DNA Fingerprinting

DNA sequence and amplification

Genetic code presence
Failed genetic response
Gene therapy
Reformatted DNA
Insulin rotation
Insulin production

PROBE Technique

Search of disabled gene

Disabled gene identification
Gene therapy

IMMUNOFLUORESCENCE Technique

Signal from DNA activation

Starting of DNA process for insulin production
Blockage of insulin production
Re-activation
Gene therapy

STERM CELL FLOW

Cell division culture

c-myc, c-fos activation/inactivation
Gene isolation
Inactivation
Gene therapy
Insulin rotation
Insulin production
CELL REGENERATION

Undead pancreatic cells

Cell culture medium + promoter drugs
Cell promotion
Medium + Mutagen drugs
c-myc and c-fos determination
Cell division
Histo-regeneration

THEORY

Fresh pancreatic cells from human organs may be sent from the Transplantation Unit.
Langerhans islets are selected from glucagon pancreatic cells. Insulin pancreatic cells are
isolated. By HPLC chromatography, the amount of insulin production is determined in
order to evaluate the severity of diabetes. The purification of pancreatic cells is carried
out by affinity chromatography for thereafter the storage. Firstly, the re-activation of
pancreatic cells in culture medium may be attempted by studying the effects of activator
drugs on sensitive pancreatic cells. According to the obtained results, we will proceed to
the identification and isolation of DNA from pancreatic cells in the purpose of
biomolecular speculations. The use of PCR technique in the amplification of DNA for the
overview of DNA sequence is required. Many copies of a specific DNA sequence can be
provide from a small initial sample (based on the natural process of DNA replication).
DNA extracted, sequenced, and compared, is used to identify. Mass-producing a DNA
sequence is called DNA amplification. PCR amplifies but does not change the initial
DNA sequence. PCR technique is done on molecules; but recombinant DNA technology
works in cells that includes sequences from other types of organisms. The use of PCR
technique will allow for the study of the genetic code presence and response to the
stimuli (activator drugs). If failed response, gene therapy for having an effective
reformatted DNA may be carry out. In culture medium, we will get some trials for insulin
production to be measured by HPLC.

Secondly, the use of a probe material for the search and identification of disabled gene is
required. This will allow for isolating and inserting a new gene in order to process to the
activation and insulin production.

Thirdly, the immunofluorescence technique may help for the signal from DNA activation
and the beginning of DNA process for insulin production. If inactivation after
reactivation of pancreatic cells, gene therapy is required. DNA might be visualized by
fluorescent in situ hybridization (FISH). FISH is a powerful molecular technique for
localized specific DNA sequences to chromosomes and regions of chromosomes.
Fluorescent in situ hybridization-treated DNA nuclei from pancreatic cells can show
normal sequences and the amplification of abnormal located on DNA in pancreatic cell
(red fluorescent signal). The green fluorescent signals are due to the hybridization of a
DNA probe specific to pancreatic cell DNA, and serve as a control.

Ultimately, fresh pancreatic samples are characterized in cell division culture. C-myc and
c-fos are determined for the capacity of these gene in cell activation and division. If
inactivation, gene therapy following the search by probe material, is required.
Nevertheless, in presence of promoter and mutagen drugs under control of using, a tissue
regeneration may be investigated.

CONCLUSION

Biomolecular methods and techniques can be used in the fundamental research and study
on diabetes type I. From using pancreatic tissue up to gene manipulations, multiple
pathways seem be affordable for a mechanism of therapeutic action against
diabetes. We hope this diabetes study design considered and applied in the future on
behalf of the knowledge and Science.

REFERENCES

(1) American College of Physicians: ACP diabetes care guide, ACP Philadelphia, 2007.

(2) Mertens TR, Hammersmith RL: Genetics laboratory investigations, Pearson

Education, San Francisco, 2007.