Methods of preventing or treating brain ischemia or brain injury
a brain injury and brain ischemia technology, applied in the field of methods, can solve the problems of brain injury, significant neurologic disability, and 200,000 deaths in the united states, and achieve the effect of preventing the effect of narp and reducing or even completely diminishing tissue damage or degeneration
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example 1
Identification and Preparation of Key Genes Involved in the Stroke Event
[0125] As a first step to the novel drug discovery, key genes involved in the stroke event were identified, as provided by the following methods:
SUMMARY OF cDNA MICRO-ARRAY CONSTRUCTION
[0126] The polynucleotide encoding Narp was found by: microarray-based differential gene expression, evaluated by both in vivo and in vitro models.
[0127] The cDNA microarray was constructed by combining cDNA libraries (Table 1), including a subtraction library, enriched for stroke specific genes. As a result, the Stroke chip consists of low-redundant stroke-specific clones. The microarray contains 10,000 cDNA clones. The libraries printed on the chip were as described in Table 1.
TABLE 1The design of Stroke chip: Library types and cDNA sources.Type ofMaterialTime pointsLibraryIn vivoIn vitro3 h6 h16 h24 hSubtraction library[MCAO]− [Sham]+L3+L4(five independent libraries)[MCAO + FK506]−+L5+L6[MCAO]Primary neurons:+L1+L1+L1+L1[...
example 2
Preparation of Taipoxin
[0156] Portions (fragments or subunits) of the toxin taipoxin may be produced via several methods, for example:
[0157] 1) Synthetically;
[0158] Synthetic polypeptides can be made using a commercially available machine, using the known sequence of the taipoxin polypeptide or fragments thereof.
[0159] 2) Recombinant Methods:
[0160] A preferred method of making the taipoxin polypeptides (preferably α,β and γ subunits) is to clone a fragment of the cDNA of the taipoxin gene into an expression vector and culture the cell harboring the vector so as to express the encoded polypeptide, and then purify the resulting polypeptide, all performed using methods known in the art (see Deutscher; Harris and Angal).
[0161] The expression vector can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription. Enhancers that can be required to obtain necessary transcription ...
example 3
[0177] The gamma subunit of taipoxin was proven to be non-toxic according to the following procedures:
[0178] A) In vitro Toxicity of Gamma-Taipoxin
[0179] P19 differentiated neurons were prepared according to protocols known in the art. Two weeks after differentiation, gamma-taipoxin or crude taipoxin are added to the cells and cell viability is subsequently measured using Alamar Blue.
[0180] Results
[0181] A concentration of 0.1 ug / ml of taipoxin caused cell death of ˜60% of P19 undifferentiated cells and ˜80% of P19 differentiated neurons. Under the selected range of concentrations, taipoxin behaves consistently, killing more than 90% of the cells.
[0182] A concentration of 1 ug / ml of gamma-taipoxin caused cell death of ˜50% of P19 differentiated neurons, while P19 undifferentiated cells were not affected by gamma-taipoxin in the test concentrations ranging from 0.5 ug / ml to 10 ug / ml.
[0183] Therefore, taking in account the molar ratio, gamma-taipoxin is several hundred ...
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